A Validated Trigonelline-Based Method for the Standardization and Quality Control of <i>Trigonella foenum-graecum</i> L.

Mohammed Aldholmi; Rizwan Ahmad; Salma Hago; Ali Alabduallah

doi:10.12688/f1000research.157659.1

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Research Article

A Validated Trigonelline-Based Method for the Standardization and Quality Control of Trigonella foenum-graecum L.

[version 1; peer review: 2 approved with reservations]

Mohammed Aldholmi¹, Rizwan Ahmad¹, Salma Hago ², Ali Alabduallah¹

PUBLISHED 11 Nov 2024

Author details Author details

¹ Department of Natural Products, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, Eastern Province, Saudi Arabia
² Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Gezira, Sudan

Mohammed Aldholmi
Roles: Conceptualization, Formal Analysis, Methodology, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Rizwan Ahmad
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Salma Hago
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ali Alabduallah
Roles: Investigation, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

Fenugreek, or Trigonella foenum-graecum L, is an edible and medicinal plant of the Fabaceae family. Fenugreek seeds contain a variety of phytochemicals, including proteins, lipids, amino acids, vitamins, flavonoids, steroidal saponins, coumarin, and alkaloids. Trigonelline TG is a bioactive plant alkaloid initially extracted from fenugreek seeds. A substantial portion of fenugreek’s health benefits may rely on the presence of TG. This study addresses the research gap for a fast, green, and economical method for quantifying trigonelline (TG) in fenugreek.

Methods

Fenugreek seeds from various origins were extracted using three green solvents: acetone (ACt), ethanol (EtOH), and water (H2O). The UPLCMSMS method was developed and validated using a green mobile phase of H2O: EtOH, and an r2-value of 0.999 in the linearity range of 0.1-500 ppb was adopted. The method was validated with an accuracy of 98.6% for trace analysis of TG using a small amount (10 mg) of fenugreek samples from five different origins.

Results

The average extract yield (±SD) was 5.36±6.3, with the highest extract yield observed in H2O. The ESI (+ve) of the UPLCMSMS resulted in the fragmentation pattern (m/z) 138→94.10→92.05→78.20. The TG quantification revealed an average TG concentration of 181.4, with the highest amount of TG in H2O extract (392.7±132.4 ppb), followed by EtOH (91.9±83.3 ppb) and ACt (59.5±30.9 ppb). The TG amount observed in the validation step substantiated the efficiency and reproducibility of the developed method.

Conclusions

The method may be used as an effective tool for a green, rapid, economical, and eco-friendly extraction and quantification of TG in diverse matrices of pharmaceutical, cosmeceutical, herbal, and food products.

Keywords

Fenugreek, UPLCMSMS, green, extraction, analysis

Corresponding author: Salma Hago

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2024 Aldholmi M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Aldholmi M, Ahmad R, Hago S and Alabduallah A. A Validated Trigonelline-Based Method for the Standardization and Quality Control of Trigonella foenum-graecum L. [version 1; peer review: 2 approved with reservations]. F1000Research 2024, 13:1350 (https://doi.org/10.12688/f1000research.157659.1) First published: 11 Nov 2024, 13:1350 (https://doi.org/10.12688/f1000research.157659.1) Latest published: 18 Dec 2024, 13:1350 (https://doi.org/10.12688/f1000research.157659.3)

Introduction

Trigonella foenum-graecum L. (fenugreek) is a medicinal and food plant that belongs to the family Fabaceae, which includes common medicinal and food plants such as Pisum sativum (pea), Cicer arietinum (chickpeas), Phaseolus (beans), Glycine max (soybean), Arachis hypogaea (peanut), and Glycyrrhiza glabra (licorice).¹ Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries.² The seed is consumed as a condiment and seasoning in food preparations and is believed to possess nutritional and health benefits.² For several centuries, fenugreek has been consumed orally as a traditional treatment for a wide range of diseases, including diabetes, fever, and abdominal colic, and applied topically for abscesses, boils, and carbuncles.³ Various pharmacological and clinical studies have confirmed significant medicinal properties of fenugreek seeds, including antidiabetic activity, anti-obesity activity, hypolipidemic activity, anticancer activity, antioxidant activity, anti-inflammatory activity, and antibacterial activity due to the presence of numerous bioactive components.⁴ Fenugreek seeds contain several classes of compounds, including proteins, lipids, amino acids, vitamins, minerals, galactomannan fibre (a carbohydrate), flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline (TG).²

TG is a bioactive plant alkaloid that was first isolated from fenugreek seeds but is also abundant in other plant-derived food products, such as coffee beans.⁵ It is an N-methylated form of nicotinic acid or vitamin B₃ (Figure 1), hence chemically known as N-methylnicotinic acid (C₇H₇NO₂). This fenugreek alkaloid has been reported to have antidiabetic, antihyperlipidemic, antimigraine, sedative, antibacterial, antiviral, anticancer, and phytoestrogenic effects; also it inhibits platelet aggregation and improves memory function.^2,4 It has been reported to demonstrate its antidiabetic efficacy by enhancing the insulin signalling pathway, alleviating endoplasmic reticulum stress, and reducing oxidative damage in individuals with type 2 diabetes.⁴ TG has been shown to enhance the axonal formation of neurons and recover memory function in Alzheimer’s disease model mice.⁶ It has also been demonstrated to incorporate into the nicotinamide adenine dinucleotide (NAD+) pool and increase NAD+ levels, improving muscle function during ageing.⁵ Therefore, this fenugreek bioactive compound appears to be a significant contributor to the medicinal properties of fenugreek seeds.

Figure 1. Chemical structures of trigonelline and nicotinic acid.

Since a significant part of the fenugreek health benefits potentially depends on the presence of TG in the utilised product, there is a critical need to develop TG-based extraction and analysis methods for the standardization and quality control of fenugreek products to ensure their efficacy and safety. The optimal extraction and analysis methods should be fast, efficient, and accurate, using an economical, eco-, and human-friendly approach. Additionally, the developed methods should avoid the use of high temperatures, as it has been reported that trigonelline breaks down to niacin at high temperatures.² Previous studies reported the use of HPLC-UV-Vis and LC-MS techniques for TG quantification, but they have the disadvantage of involving non-green and toxic solvents in the mobile phase system.^7–12 Therefore, a loophole still exists for the extraction and determination of TG in fenugreek samples using a fast, green, economical, eco-friendly, and less toxic methodology. The current study aims to investigate the efficiency of three green solvents for extraction of TG from fenugreek using an ultrasonic dismembrator (20 kHz), develop an economical, eco-, and human-friendly analysis method, and practically apply the method to fenugreek samples from different origins.

Methods

Solvents, chemicals, and instruments

Analytical-grade acetone (ACt, Cat. 107021) and ethanol (EtOH, Cat. 107017), as well as HPLC-grade ethanol (EtOH, Cat. 111727), were purchased from Merck (Darmstadt, Germany). Water (H₂O) used for extraction and LCMSMS analysis was produced in-house using Millipore equipment (Millipore, Bedford, MA, USA). The standard chemical trigonelline (TG, Cat. 1686411) was obtained from Sigma Aldrich St Louis, MO, USA.

Fisher Scientific (2000 Park Lane Pittsburgh, PA, USA) ultrasonics dismembrator [power (50 Watt), frequency (20 kHz), transducer (Model CL-334), horn (220 A), titanium probe (420 A; 1 mm diameter)] was used for extraction. Ultra-pressure liquid chromatography-mass spectrometry (UPLC-MS/MS) instrument (Shimadzu, Japan, LCMS-8050) consisting of binary pumps (LC-30AD), thermostatted column compartment (CTO30A), UV-DAD detector (SPD-M20A), and triple-quadrupole mass detector with electrospray-ionisation-source (TQMS-ESI) was used for quantitative analysis. The data was processed and analysed with the help of LabSolutions software (Kyoto, Japan V 5.93). The evaporation and drying of the solvents from the samples were accomplished with the help of a miVac Quattro Concentration System (Genevac Ltd., Ipswich, United Kingdom). The analytical column used for the separation was a C18 Acquity UPLC column from Waters (2.1 × 100 mm; 1.7 mm).

Fenugreek samples collection and preparation

Dried fenugreek seed samples originating from five different countries consisting of India, Egypt, Iran, Saudi Arabia, and Yemen were collected from local markets in the Kingdom of Saudi Arabia, as reported in our previous study.¹³ Three different types of fenugreek seeds (based on color and size) were collected for each origin, resulting in fifteen fenugreek samples (3 × 5 = 15).

Green Ultrasonics Extraction (USE) for TG

Fenugreek seed samples were extracted for the three accessions from each origin using three green solvents of ACt, EtOH, and H₂O, resulting in forty-five fenugreek extracts (3 × 5 × 3 = 45). The samples were properly coded from F1 to F15 plus A, B, and C letters standing for ACt, EtOH, and H₂O, respectively. The extraction procedure was adopted with slight modification from the previously developed in-house USE method.¹⁴ Briefly, 100 mg of the fenugreek seeds from each accession were weighed, and 10 mL of the respective solvent was added. The amplitude and pulse were set at 30% and 30/10 s, respectively. The five-minute extraction was followed by the evaporation of solvents using the miVac Quattro Concentration System (Genevac Ltd., Ipswich, United Kingdom). The dried samples were weighed for extract yield calculation (mg/100 mg).

Green UPLCMSMS analytical method for TG

Method development for green UPLCMSMS analysis

The standard drug TG was dissolved (1 mg/mL) in HPLC-grade EtOH to prepare a stock solution. For working standards, the stock solution was further diluted in the required solvent volume to construct six points in the linearity range of 0.1-500 ppb (0.1, 5, 10, 50, 100, and 500 ppb). The extracts, stock, and working standard solutions were prepared in HPLC-grade EtOH and filtered through a syringe filter (0.2 mm). For separation and quantitation of TG, the mobile phase of H₂O (A) and EtOH (B) was used to run the TG standard solutions with a gradient of 5-95% B using the C18 column. Mobile phase at different gradient and isocratic elution compositions, flow rates (0.1, 0.2, 0.3 mL/min), and injection volumes (1, 2, 5 mL) were tested for the efficient separation of TG.

The conditions used for mass spectrometry were optimised to obtain the most appropriate ionisation and fragmentation pattern. The ESI (electrospray ionisation) interface was used for the mass analysis, and a positive MRM (multiple reaction monitoring) mode was applied to quantify and analyse the daughter fragments using an external standard calibration curve. Following the successful fragmentation via optimal collision energy (CE) selection, the fragment (m/z) with the highest intensity (base peak), along with the retention time (Rt) of the peak, were used to identify the target analyte in all samples. The final LC and MS conditions are presented in Section Chromatographic separation and MS optimisation.

Method validation for green UPLCMSMS analysis

The UPLCMSMS-MD was validated in terms of accuracy, linearity, LOD (limit of detection), and LOQ (limit of quantification). The linearity of the method was evaluated using six different calibration points in the range of 0.1-500 ppb, where the regression equation and r²-value were calculated. The accuracy, LOD, and LOQ were calculated via in-built LabSolutions software using the ICH guidelines¹⁵ and previously validated parameters for the UPLCMSMS analytical method.¹⁶

Applicability of the USE-UPLCMSMS for trace analysis

The developed USE-UPLCMSMS was further tested in terms of efficiency and applicability of the method for trace analysis by H₂O extraction and analysis of lower amounts (10 mg) of the fenugreek samples containing the lowest TG quantity from each origin (F1, F5, F8, F10, and F15), as mentioned previously. The extracted samples were dried, diluted, and prepared for UPLCMSMS analysis to determine TG concentrations.

Results

Extracts yield

Determination of the extract yield in different fenugreek samples

The extract yield for the forty-five fenugreek extracts (N=45) exhibited a sum of 241.0 mg/4500 mg with a mean (±SD) of 5.36±6.3. The range for the extract yield observed was (minimum to maximum) 1.1-30.6 mg (N=45). For the average extract yield/origin (N=9: mg/100 mg), the descending order observed was Egypt (7.1 ±8.5 mg)> Yemen (6.6±9.2 mg)> Saudi Arabia (6.0±6.0 mg)> Iran (3.7±2.9 mg)> India (3.4±2.0 mg). With regard to the highest extract yield in individual fenugreek samples of each origin (N=9), the following samples exhibited the highest extract yield in each origin: F3C in H₂O (6.3 mg) for the Indian samples, F5C in H₂O (21.5 mg) for the Saudi Arabian, F7A in ACt (9.6 mg) for the Iranian, F11C in H₂O (28.1 mg) for the Egyptian, and F15C in H₂O (30.6 mg) for the Yemeni origin. The fenugreek samples noted with the highest yield among the 45 samples were F15C (30.6 mg)> F11C (28.1 mg)> F5C (21.5 mg)> F11A (10.8 mg). The results of average and individual yields in different origins are summarized in Table 1.

Table 1. Average and individual extract yields for fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B), and H₂O (C).

Country of origin	Sample code	Extract yield (mg/100 mg)	Average Yield/origin (mg/100 mg)	SD
India	F1A	5.6	3.4	2.0
	F1B	1.3
	F1C	1.1
	F2A	5.3
	F2B	2.1
	F2C	4.2
	F3A	3.0
	F3B	1.8
	F3C	6.3
Saudi Arabia	F4A	4.3	6.0	6.0
	F4B	3.7
	F4C	6.0
	F5A	3.4
	F5B	1.8
	F5C	21.5
	F6A	5.9
	F6B	2.2
	F6C	5.5
Iran	F7A	9.6	3.7	2.9
	F7B	1.4
	F7C	2.9
	F8A	7.5
	F8B	1.7
	F8C	3.8
	F9A	3.1
	F9B	1.1
	F9C	2.1
Egypt	F10A	7.6	7.1	8.5
	F10B	1.4
	F10C	4.9
	F11A	10.8
	F11B	4.2
	F11C	28.1
	F12A	3.1
	F12B	1.1
	F12C	2.4
Yemen	F13A	3.2	6.6	9.2
	F13B	1.2
	F13C	3.9
	F14A	3.6
	F14B	1.4
	F14C	4.2
	F15A	7.5
	F15B	3.6
	F15C	30.6

Variation of extract yield in green solvents

For the green solvents, the sum and mean of extract yield in each solvent (N=15) were 83.5 and 5.6 mg for ACt, 30.0 mg and 2.0 mg for EtOH, and 127.5 and 8.5 for H₂O, respectively. The average extract yield for each individual origin in the extraction solvents resulted in the following descending order: India (4.6±1.4 mg (ACt)> 3.9±2.6 mg (H₂O)> 1.7±0.4 mg (EtOH); Saudi Arabia (11.0±9.1 (H₂O)> 4.5±1.3mg (ACt)> 2.6±1.0 mg (EtOH); Iran (6.7±3.3 mg (ACt)> 2.9±0.9 mg (H₂O)> 1.4±0.3 mg (EtOH); Egypt (11.8±14.2 mg (H₂O)> 7.2±3.9 mg (ACt)> 2.2±1.7 mg (EtOH)); and Yemen (12.9±15.3 mg (H₂O)> 4.8±2.4 mg (ACt)> 2.1±1.3 mg (EtOH).

On an individual basis, the highest extract yield was observed for H₂O (30.6 mg/100 mg) in the Yemeni fenugreek sample, followed by ACt (10.8 mg/100 mg) in the Egyptian fenugreek sample, and EtOH (3.7 mg/100 mg) in the Saudi Arabian fenugreek sample. The total and average extract yield data for the fenugreek samples suggest a descending order of extract yield with the superior solvent to be H₂O>ACt> EtOH. The data for the extract yield in green solvents is shown in detail in Table 2.

Table 2. Yield/Origin (mg) and total extract yield (mg) in green solvents for fenugreek samples with SD.

Origin	Code	Yield in Act (A)			Yield in EtOH (B)			Yield in H₂O (C)
Origin	Code	Yield/sample	Average/Origin	SD	Yield/sample	Average/Origin	SD	Yield/sample	Average/Origin	SD
India	F1	5.6	4.6	1.4	1.3	1.7	0.4	1.1	3.9	2.6
	F2	5.3			2.1			4.2
	F3	3.0			1.8			6.3
Saudi Arabia	F4	4.3	4.5	1.3	3.7	2.6	1.0	6.0	11.0	9.1
	F5	3.4			1.8			21.5
	F6	5.9			2.2			5.5
Iran	F7	9.6	6.7	3.3	1.4	1.4	0.3	2.9	2.9	0.9
	F8	7.5			1.7			3.8
	F9	3.1			1.1			2.1
Egypt	F10	7.6	7.2	3.9	1.4	2.2	1.7	4.9	11.8	14.2
	F11	10.8			4.2			28.1
	F12	3.1			1.1			2.4
Yemen	F13	3.2	4.8	2.4	1.2	2.1	1.3	3.9	12.9	15.3
	F14	3.6			1.4			4.2
	F15	7.5			3.6			30.6
Sum	83.5				30.0			127.5
Mean	5.6				2.0			8.5
SD	2.5				1.0			9.7

Chromatographic separation and MS optimisation

The chromatographic method development resulted in a mobile phase of H₂O (A)+EtOH (B) with an isocratic elution of 30 (A):70 (B). The retention time for TG was 0.882 min with a runtime of 1.5 min at a flow rate of 0.3 mL/min. The method was validated using ICH guidelines in the linearity range of 0.1-500 ppb, where an accuracy of 98.6% with an r²-value of 0.999 was obtained, showing the efficiency and reproducibility of the method. For MS optimisation, a positive (+ve) MRM mode with an ESI interface resulted in the daughter fragments (m/z) of 138 →94.10 →92.05 →78.20. The base peak of 94.10 was selected for the data analysis and calibration curve development using the six different concentrations of calibration points. The details regarding LC separation and MS optimisation are presented in Table 3. A representative chromatogram showing the Rt for the TG-peak is given in Figure 2 whereas the mass fragmentation pattern with the base peak for TG is shown in Figure 3. The details regarding the calibration curve, regression equation, and r²-value with the linearity range points are presented in Figure 4.

Table 3. LC and MS conditions for the analysis of TG in fenugreek samples.

LC and MS parameters
Mobile phase			H₂O (A)+EtOH (B)
Mobile phase composition			Isocratic at 30 (A):70 (B)
Flow rate (FR)			0.3 mL/min
Rt (retention time)			0.882 min
Runtime			1.5 min
LOD (limits of detection)			0.5 ppb
LOQ (limits of quantification)			1.4 ppb
Accuracy			98.6%
Regression equation			Y=22846.9x+100685
r²-value			0.999
Linearity range			0.1-500 ppb
TG-fragmentation pattern	Precursor mass	Product m/z	Q1 pre-bias (V)	CE	Q3 pre-bias (V)
	138	94.10	-12.0	-22.0	-17.0
	138	92.05	-10.0	-20.0	-22.0
	138	78.20	-13.0	-25.0	-14.0
MRM mode			+ve
Dwell time			100 msec
Event time			0.309
Mass end time			1.5
Interface			ESI
Nebulizing gas flow			3 L/min
Drying gas flow			10 L/min
Heating gas flow			10 L/min
Interface temperature			300°C
DL temperature			250°C
Heat block temperature			400°C
Interface current			0.4 uA

Figure 2. UPLCMSMS chromatogram of TG standard.

Figure 3. MSMS fragmentation pattern of TG (m/z).

Figure 4. Standard calibration curve of TG.

TG quantification

UPLCMSMS analysis of TG in different fenugreek samples

The TG amount observed was within the range of (minimum to maximum) 22.2-535.0 ppb (N = 45). The sum and mean (±SD) for the TG amount in the fenugreek samples (N = 45) were 8161 ppb and 181.4±176.4 ppb, respectively. The average TG amount/individual origin (N=9; ppb) created a descending order of Yemen (220.0±191.7)> India (211.3±198.1)> Saudi Arabia (190.8±210.7)> Egypt (189.5±165.2)> Iran (95.2±110.4). The greatest TG amounts were observed in the following fenugreek samples: 535.0 ppb for F3C (Indian)> 510.0 ppb for F6C (Saudi Arabian)> 491.0 ppb for F4C (Saudi Arabian)> 475.0 ppb for F14C (Yemeni).

For the highest TG amount in each individual origin (N = 9), an amount of 535.0 ppb in H₂O was observed for F3C (India), 510.0 ppb in H₂O for F6C (Saudi Arabia), 370.0 ppb in H₂O for F9C (Iran), 423.0 ppb in H₂O for F12C (Egypt), and 475.0 ppb in H₂O for F14C (Yemen). Detailed data regarding the TG amount in 45 different samples of fenugreek seeds is provided in Table 4.

Table 4. Average and individual TG amounts in fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B) and H₂O (C).

Country of origin	Sample code	TG amount (ppb)	Average TG amount/origin (ppb)	SD
India	F1A	56.6	211.3	198.1
	F1B	105.5
	F1C	434.0
	F2A	43.2
	F2B	146.0
	F2C	437.0
	F3A	36.7
	F3B	108.1
	F3C	535.0
Saudi Arabia	F4A	37.0	190.8	210.7
	F4B	46.7
	F4C	491.0
	F5A	80.4
	F5B	62.3
	F5C	405.0
	F6A	34.3
	F6B	50.1
	F6C	510.0
Iran	F7A	27.3	95.2	110.4
	F7B	22.2
	F7C	92.6
	F8A	22.6
	F8B	24.3
	F8C	82.9
	F9A	133.2
	F9B	81.7
	F9C	370.0
Egypt	F10A	100.7	189.5	165.2
	F10B	107.5
	F10C	391.0
	F11A	47.2
	F11B	35.4
	F11C	407.0
	F12A	91.6
	F12B	102.3
	F12C	423.0
Yemen	F13A	49.1	220.0	191.7
	F13B	102.7
	F13C	452.0
	F14A	74.3
	F14B	359.2
	F14C	475.0
	F15A	58.4
	F15B	24.4
	F15C	385.0

Variation of TG amount in green solvents

The UPLCMSMS analysis of the green extracts of fenugreek seeds exhibited the highest average amount of TG (N=15) in H₂O (392.7±132.4 ppb) followed by EtOH (91.9±83.3 ppb) and ACt (59.5±30.9 ppb). On an individual basis, the highest amount of TG was detected in H₂O extracts of all fenugreek samples (N=45) when compared to the EtOH and ACt extracts of the same samples.

For the average TG amount in the three solvent extracts of each individual origin, a descending order may be constructed (N=9) as follows: India (468.7±57.5 ppb (H₂O)> 119.9±22.7 ppb (EtOH)> 45.5±10.1 ppb (ACt); Saudi Arabia (468.7±55.9 ppb (H₂O)> 53.0±8.2 ppb (EtOH)> 50.6±25.9 ppb (ACt); Iran (181.8±163.0 ppb (H₂O)> 61.0±62.6 ppb (ACt)> 42.7±33.8 ppb (EtOH); Egypt (407.0±16.0 ppb (H₂O)> 81.7±40.2 ppb (EtOH)> 79.8±28.6 ppb (ACt); and Yemen (437.3±46.8 ppb (H₂O)> 162.1±175.1 ppb (EtOH)> 60.6±12.8 ppb (ACT). The average and individual TG amounts suggest the descending order for solvents with optimal TG amount to be H₂O> EtOH> ACt. The data regarding the TG amount is provided in detail in Table 5.

Table 5. TG amount/Origin (ppb) and total TG amount (ppb) quantified via UPLCMSMS in the green extracts of fenugreek.

Origin	Code	TG in Act (A)			TG in EtOH (B)			TG in H₂O (C)
Origin	Code	TG/sample	Average/Origin	SD	TG/sample	Average/Origin	SD	TG/sample	Average/Origin	SD
India	F1	56.6	45.5	10.1	105.5	119.9	22.7	434.0	468.7	57.5
	F2	43.2			146.0			437.0
	F3	36.7			108.1			535.0
Saudi Arabia	F4	37.0	50.6	25.9	46.7	53.0	8.2	491.0	468.7	55.9
	F5	80.4			62.3			405.0
	F6	34.3			50.1			510.0
Iran	F7	27.3	61.0	62.6	22.2	42.7	33.8	92.6	181.8	163.0
	F8	22.6			24.3			82.9
	F9	133.2			81.7			370.0
Egypt	F10	100.7	79.8	28.6	107.5	81.7	40.2	391.0	407.0	16.0
	F11	47.2			35.4			407.0
	F12	91.6			102.3			423.0
Yemen	F13	49.1	60.6	12.8	102.7	162.1	175.1	452.0	437.3	46.8
	F14	74.3			359.2			475.0
	F15	58.4			24.4			385.0
Sum	892.6				1378.4			5890.5
Mean	59.5				91.9			392.7
SD	30.9				83.3			132.4

Applicability of USE-UPLCMSMS for trace analysis

The developed USE-UPLCMSMS was further tested in terms of the efficiency and applicability of the method for trace analysis by H₂O extraction and analysis of lower amounts (10 mg) of fenugreek samples. A sample from each origin (F1 for India, F5 for Saudi Arabia, F8 for Iran, F10 for Egypt, and F15 for Yemen) was selected for this purpose. The developed USE-UPLCMSMS was able to extract and detect TG in the low-amount samples (10 mg) with a minimum amount of 20 ppb in F8 (Iran). The TG amount from the 10 mg extracts of fenugreek samples (N=5) showed a sum of 403.4 ppb with a mean (±SD) of 80.68±34.28 ppb. The TG amount ranged from a minimum of 20 ppb to a maximum amount of 103.5 ppb. The data for the TG amounts in all five samples is presented in Figure 5.

Figure 5. Trace analysis of TG in 10 mg of five fenugreek samples.

Discussion

There is an enormous trend for the application of fenugreek seeds in soaps, cosmetics, herbals, and dietary supplements due to their nutraceutical and medicinal values. Additionally, fenugreek seeds are used to enhance the sensory quality of food and spices.¹⁷ Hence, there is an utmost need for a methodology to extract and analyse TG in a sample with an economical, eco-, and human-friendly approach at the same time. This study focuses on the development and validation of a green, fast, efficient, and reproducible method for TG extraction and analysis with the potential to effectively evaluate the quality of different fenugreek seeds in terms of their TG content. The researchers herein strive to develop an economical, eco-, and human-friendly analytical method. In this regard, fifteen fenugreek seeds from different origins were collected from the local markets of Riyadh and Jeddah, Saudi Arabia. The uncrushed seeds, without any pretreatment, were weighed and subjected to extraction using three different green solvents: ACt, EtOH, and H₂O. The extraction technique used was an ultrasonic dismembrator with high-frequency ultrasonic waves (20 kHz). The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time.^14,16 The USE-MD used three green solvents (ACt, EtOH, and H₂O) to assess the optimal solvent with the highest TG amount. The extraction results, represented by the individual and total extract yield, suggested H₂O as an appropriate solvent for extraction.

This study reports a high aqueous extract yield and TG amount for H2O among the three green solvents, which agrees with previous studies reporting H2O as an efficient extraction medium for TG.^7,8 The total extract yield for the green solvents exhibited an order of H2O>ACt>EtOH. Moreover, the highest yield on an individual basis was observed for H2O in the Yemeni fenugreek sample. The aim of this study was to quantify the TG amount in these fenugreek samples; hence, an advanced analytical instrument of UPLCMSMS was employed to develop and validate a green in-house method for TG quantification. A rapid, sensitive, green, and reproducible method was developed using a green mobile phase of H2O and EtOH with a low flow rate of 0.3 mL/min in a short runtime of 1.5 min with equilibration. The developed method was validated for accuracy, detection and quantification limits, and coefficient value determination as per ICH guidelines, where all the results were obtained within the specified limits. To the best of our knowledge, this is the first green, sensitive, and rapid UPLC-MSMS analysis method for the quantification of TG in fenugreek samples. Previous studies reported the use of HPLC-UV-Vis and LC-MS techniques for TG quantification in coffee beans, fenugreek, and dietary supplements available in the market, but non-green and toxic solvents were used in the mobile phase system.^7–10 Therefore, the main strength of this study is the use of green solvents for extraction and analysis of TG in fenugreek samples. A successful green and rapid extraction and analysis method was developed in this study.

Following a successful UPLCMSMS, the extracted samples were analysed using the in-house method. A similar pattern was observed for the extracted samples, where the highest TG amounts were observed in the H2O samples. A descending order for the solvent with the highest quantities was constructed for all the forty-five extracted samples as H2O> EtOH> ACt. Previous studies have reported trigonelline to be freely soluble in water.^7–12 Therefore, the solubility and extraction behaviour for TG in the aqueous medium complement the concept of TG aqueous solubility, which corroborates the outcomes.

This is the first time to report a comprehensive assessment of TG in fifteen samples from different origins marketed in Saudi Arabia using a green, rapid, efficient, and reliable method of USE-UPLCMSMS. The method assessed the quality of the fenugreek samples in terms of their TG concentration, where significant variation was observed among the samples from different origins.

The real challenge is to validate the efficiency and reproducibility of the developed USE-UPLCMSMS method to be applied practically for the assessment and evaluation of low concentrations of target analytes in complex matrices. At times, the challenge of trace analysis for TG in samples of dietary supplements or marketed products may be encountered; hence, it becomes essential to authenticate the scope of the developed method for quantification of TG at low amounts in a matrix. To prepare the method for the mentioned challenge, an extra validation step was undertaken for extraction and trace analysis of TG. A small amount (10x lower in concentration) of the fenugreek amount for the five different origin samples was weighed and extracted with USE as previously described. The extracted samples were diluted and analyzed via the UPLCMSMS analytical method. The quantification of TG was successfully accomplished at the lowest TG concentration present in the tested fenugreek samples, ensuring the applicability and reliability of the developed method.

The USE-UPLCMSMS method developed in this study extends the benefit of using TG as a marker for the quality control and standardization of any pharmaceutical, cosmeceutical, herbal, and dietary products containing TG. The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes. Another add-on advantage is the use of H₂O as a medium for extraction, which is usually produced within the labs using Millipore apparatus. Overall, this method offers high levels of convenience, reliability, sensitivity, and accuracy for the quality evaluation and standardization of any TG-containing samples.

Conclusions

The study developed a green, fast, economical, and reliable method (USE-UHPLCMSMS) for the extraction and analysis of TG in different fenugreek seed extracts collected from different geographical origins. The method was further validated to ensure the efficiency in matrices containing trace amounts of TG. The use of water with the lowest possible amount of samples is considered a superior economical point of the developed extraction method, in addition to the green and short analysis time of the UPLCMSMS analytical method. The USE-UPLCMSMS method may serve as a tool in research labs and for the quality control and standardization of TG amounts in different commercial herbal products and dietary supplements.

Ethics and consent

The present study did not involve the use of human or animals; therefore, ethical approval and consent were not required.

Authors’ contributions

Conceptualization, S.H., M.A., and R.A.; Methodology, M.A., R.A. and S.H.; Software, M.A. and R.A.; Validation, M.A., S.H. and R.A.; Formal Analysis, M.A. and R.A.; Investigation, A.A. and R.A.; Resources, M.A. and R.A.; Data Curation, S.H. and R.A.; Writing—Original Draft Preparation, M.A., R.A., A.A. and S.H.; Writing—Review and Editing, M.A., R.A., S.H. and A.A.; Visualization, M.A., S.H. and R.A.; Supervision, S.H. and R.A.; Project Administration, S.H. and R.A. All authors have read and agreed to the final version of the manuscript.

Data and software availability

Underlying data

Zenodo: A validated trigonelline-based method for the standardization and quality control of Trigonella foenum-graecum L., https://doi.org/10.5281/zenodo.13991270.¹⁸

This project contains the following underlying data:

• Raw data for extract yields and trigonelline amounts in fenugreek (extract yields and trigonelline amounts in 45 fenugreek samples).
• Trigonelline LC-MSMS data (mass spectrum of trigonelline).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Software availability statement

LabSolutions is proprietary software used for the analysis of mass spectra and the calculation of method validation parameters, such as the regression equation, coefficient of determination (R²), accuracy, linearity, limit of detection (LOD), and limit of quantification (LOQ). The mass spectral data analysis and method validation calculations presented in this article, originally performed with LabSolutions, can alternatively be conducted using the freely accessible software LibreOffice version 24.8.2.

License: OSI approved open license software is under the Mozilla Public License (MPL) 2.0.

Acknowledgements

We would like to acknowledge the technicians in the natural products department, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, for their assistance in the project.

References

1. Family Fabaceae: Vegetable Seeds. CABI: GB; 2024; pp. 236–259. Publisher Full Text
2. Garg RC: Fenugreek. Nutraceuticals. Elsevier; 2016; pp. 599–617. Publisher Full Text
3. Zhou J, Chan L, Zhou S: Trigonelline: A Plant Alkaloid with Therapeutic Potential for Diabetes and Central Nervous System Disease. Curr. Med. Chem. 2012; 19: 3523–3531. PubMed Abstract | Publisher Full Text
4. Faisal Z, Irfan R, Akram N, et al.: The multifaceted potential of fenugreek seeds: From health benefits to food and nanotechnology applications. Food Sci. Nutr. 2024; 12: 2294–2310. PubMed Abstract | Publisher Full Text | Free Full Text
5. Membrez M, Migliavacca E, Christen S, et al.: Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia. Nat. Metab. 2024; 6: 433–447. PubMed Abstract | Publisher Full Text | Free Full Text
6. Farid MM, Yang X, Kuboyama T, et al.: Trigonelline recovers memory function in Alzheimer’s disease model mice: evidence of brain penetration and target molecule. Sci. Rep. 2020; 10: 16424. PubMed Abstract | Publisher Full Text | Free Full Text
7. Liu H, Li Q, Shao J, et al.: Determination of trigonelline in coffee powder and instant coffee by ultrasonic extraction and high performance liquid chromatography. Se pu Chinese J. Chromatogr. 2011; 29: 1103–1106.
8. Atlabachew M, Abebe A, Alemneh T, et al.: Rapid and simultaneous determination of trigonelline, caffeine, and chlorogenic acid in green coffee bean extract. Food Sci. Nutr. 2021; 9: 5028–5035. PubMed Abstract | Publisher Full Text | Free Full Text
9. Al-Juhaimi F, Adiamo OQ, Ghafoor K, et al.: Optimization of ultrasonic-assisted extraction of phenolic compounds from fenugreek (Trigonella foenum-graecum L.) seed. CyTA - J. Food. 2015; 14(3): 369–374. Publisher Full Text
10. Zhai X, Yang M, Zhang J, et al.: Feasibility of Ultrasound-Assisted Extraction for Accelerated Cold Brew Coffee Processing: Characterization and Comparison With Conventional Brewing Methods. Front. Nutr. 2022; 9: 9. PubMed Abstract | Publisher Full Text | Free Full Text
11. Singh P, Bajpai V, Gond V, et al.: Determination of Bioactive Compounds of Fenugreek (Trigonella foenum-graecum) Seeds Using LC-MS Techniques. Methods Mol. Biol. 2107; 377–393. PubMed Abstract | Publisher Full Text
12. Wadhwa G, Krishna KV, Taliyan R, et al.: A novel UPLC–MS/MS method for simultaneous quantification of trigonelline, 4-hydroxyisoleucine, and diosgenin from Trigonella foenum-graecum extract: Application to pharmacokinetic study in healthy and type 2 diabetic rats. Biomed. Chromatogr. 2022; 36: e5275. PubMed Abstract | Publisher Full Text
13. Ahmad R, Alqathama A, Aldholmi M, et al.: Ultrasonic-assisted extraction of fenugreek flavonoids and its geographical-based comparative evaluation using green UHPLC-DAD analysis. Ultrason. Sonochem. 2023; 95: 106382. PubMed Abstract | Publisher Full Text | Free Full Text
14. Ahmad R, Aldholmi M, Alqathama A, et al.: Green and novel ultrasonic extraction with UHPLC-MSMS analysis of natural sweetener (Glycyrrhizic acid) from Glycyrrhiza glabra; a multifactorial mechanistic evaluation based on statistical analysis. Ultrason. Sonochem. 2021; 77: 105696. PubMed Abstract | Publisher Full Text | Free Full Text
15. International conference on harmonisation of technical requirement for registration of pharmaceuticals for human use Ich Harmonised Tripartite Guideline Validation of Analytical P Rocedures.1994.
16. Ahmad R, Aldholmi M, Alqathama A, et al.: The effect of natural antioxidants, pH, and green solvents upon catechins stability during ultrasonic extraction from green tea leaves (Camellia sinensis). Ultrason. Sonochem. 2023; 94: 106337. PubMed Abstract | Publisher Full Text | Free Full Text
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18. Aldholmi M, Ahmad R, Hago S, et al.: A validated trigonelline-based method for the standardization and quality control of Trigonella foenum-graecum L. [Dataset]. Zenodo. 2024. Publisher Full Text

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Version 3

VERSION 3 PUBLISHED 11 Nov 2024

Author details Author details

Mohammed Aldholmi
Roles: Conceptualization, Formal Analysis, Methodology, Resources, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Ali Alabduallah
Roles: Investigation, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (3)

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Published: 18 Dec 2024, 13:1350

https://doi.org/10.12688/f1000research.157659.3

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Published: 11 Nov 2024, 13:1350

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© 2024 Aldholmi M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Aldholmi M, Ahmad R, Hago S and Alabduallah A. A Validated Trigonelline-Based Method for the Standardization and Quality Control of Trigonella foenum-graecum L. [version 1; peer review: 2 approved with reservations]. F1000Research 2024, 13:1350 (https://doi.org/10.12688/f1000research.157659.1)

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Open Peer Review

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Reviewer Report 26 Nov 2024

Abdul Sadiq, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan

Approved with Reservations

https://doi.org/10.5256/f1000research.173145.r340136

The manuscript presents a scientifically sound and innovative method for the extraction and analysis of trigonelline in fenugreek seeds. The approach is environmentally sustainable, reproducible, and suitable for widespread application. Minor enhancements could be considered for future work, but the study meets the standards for publication. However, there are a few minor areas where clarity or wording could be slightly improved for better readability:

Abstract:
"Fenugreek seeds contain a variety of phytochemicals, including proteins, lipids, amino acids, vitamins, flavonoids, steroidal saponins, coumarin, and alkaloids."
Phytochemicals usually refer to biologically active compounds produced by plants, which usually include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are being mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be:
"Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Introduction:
"Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries."
The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.

Methodology:
"The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time."
The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."

Conclusion:
"The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes."
Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Pharmaceutical Sciences

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

04 Dec 2024

Author Response

Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins ... Continue reading Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."
Response: We agree with the reviewer’s comment regarding the definition of phytochemicals. The sentence in the abstract has been revised to separate proteins and amino acids from the phytochemical category for accuracy. The updated sentence now reads: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Reviewer Comment 2: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries." The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.
Response: We appreciate the reviewer’s suggestion to avoid redundancy. The sentence has been revised to remove "and other countries" for conciseness. It now reads: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North Africa, and India."

Reviewer Comment 3: "The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time." The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."
Response: We agree with the reviewer’s suggestion to simplify this sentence. The revised sentence now reads: "The USE protocol was selected due to the numerous advantages of USE."

Reviewer Comment 4: "The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes." Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.
Response: We appreciate this suggestion to enhance clarity. The sentence has been split into two, as follows: "The method avoids the need for additional steps, such as seed crushing and sieving, by using an ultrasonic dismembrator. Furthermore, the method employs small sample amounts (10 mg) and low solvent volumes, ensuring cost-efficiency and eco-friendliness."
Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."
Response: We agree with the reviewer’s comment regarding the definition of phytochemicals. The sentence in the abstract has been revised to separate proteins and amino acids from the phytochemical category for accuracy. The updated sentence now reads: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Reviewer Comment 2: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries." The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.
Response: We appreciate the reviewer’s suggestion to avoid redundancy. The sentence has been revised to remove "and other countries" for conciseness. It now reads: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North Africa, and India."

Reviewer Comment 3: "The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time." The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."
Response: We agree with the reviewer’s suggestion to simplify this sentence. The revised sentence now reads: "The USE protocol was selected due to the numerous advantages of USE."

Reviewer Comment 4: "The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes." Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.
Response: We appreciate this suggestion to enhance clarity. The sentence has been split into two, as follows: "The method avoids the need for additional steps, such as seed crushing and sieving, by using an ultrasonic dismembrator. Furthermore, the method employs small sample amounts (10 mg) and low solvent volumes, ensuring cost-efficiency and eco-friendliness."
Competing Interests: No competing interest Close
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Author Response 04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

04 Dec 2024

Author Response

Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins ... Continue reading Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."
Response: We agree with the reviewer’s comment regarding the definition of phytochemicals. The sentence in the abstract has been revised to separate proteins and amino acids from the phytochemical category for accuracy. The updated sentence now reads: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Reviewer Comment 2: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries." The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.
Response: We appreciate the reviewer’s suggestion to avoid redundancy. The sentence has been revised to remove "and other countries" for conciseness. It now reads: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North Africa, and India."

Reviewer Comment 3: "The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time." The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."
Response: We agree with the reviewer’s suggestion to simplify this sentence. The revised sentence now reads: "The USE protocol was selected due to the numerous advantages of USE."

Reviewer Comment 4: "The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes." Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.
Response: We appreciate this suggestion to enhance clarity. The sentence has been split into two, as follows: "The method avoids the need for additional steps, such as seed crushing and sieving, by using an ultrasonic dismembrator. Furthermore, the method employs small sample amounts (10 mg) and low solvent volumes, ensuring cost-efficiency and eco-friendliness."
Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."
Response: We agree with the reviewer’s comment regarding the definition of phytochemicals. The sentence in the abstract has been revised to separate proteins and amino acids from the phytochemical category for accuracy. The updated sentence now reads: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Reviewer Comment 2: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries." The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.
Response: We appreciate the reviewer’s suggestion to avoid redundancy. The sentence has been revised to remove "and other countries" for conciseness. It now reads: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North Africa, and India."

Reviewer Comment 3: "The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time." The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."
Response: We agree with the reviewer’s suggestion to simplify this sentence. The revised sentence now reads: "The USE protocol was selected due to the numerous advantages of USE."

Reviewer Comment 4: "The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes." Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.
Response: We appreciate this suggestion to enhance clarity. The sentence has been split into two, as follows: "The method avoids the need for additional steps, such as seed crushing and sieving, by using an ultrasonic dismembrator. Furthermore, the method employs small sample amounts (10 mg) and low solvent volumes, ensuring cost-efficiency and eco-friendliness."
Competing Interests: No competing interest Close
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Reviewer Report 22 Nov 2024

Abdulrahman Koshak, King Abdulaziz University, Jeddah, Saudi Arabia

Approved with Reservations

https://doi.org/10.5256/f1000research.173145.r340140

The article presents a novel analytical technique for quantifying the marker compound TG in fenugreek using UPLC-MS/MS. However, it would benefit from a more comprehensive discussion of the disadvantages associated with traditional methods in comparison to the developed approach. For example, elaborating on the advantages of UPLC-MS/MS, the types of solvents commonly used in traditional methods, their potential drawbacks, and how the new method overcomes these challenges would help to better clarify the research gap.

Title: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study.

Abstract:

Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
In the results section, include the units for extract yield and TG concentration for clarity.

Formatting:

Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.

Results:

Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.

Discussion:

The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Pharmacognosy and natural products

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Report a concern

Author Response 04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

04 Dec 2024

Author Response

Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain ... Continue reading Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain the current title because the study has already been published. Changing the title could cause confusion and inconsistency in citations and references to the work. However, we have ensured that the terms "green" and "UPLC-MS/MS" are included in the "Keywords" section to emphasize the innovative and advantageous aspects of the study.

Abstract Reviewer Comment 1: Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Response: The abstract has been revised to better articulate the research gap and the limitations of traditional methods, including their inefficiency, reliance on high solvent volumes, and lack of sensitivity.

Reviewer Comment 2: Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
Response: The brackets around "(TG)" have been removed.

Reviewer Comment 3: Include the units for extract yield and TG concentration in the results section for clarity.
Response: Units for extract yield (mg/100mg) and TG concentration (ppb) have been added to the results section of the abstract.

Formatting Reviewer Comment 4: Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Response: The manuscript has been reviewed, and we have ensured that "UPLC-MS/MS" is consistently formatted throughout.

Reviewer Comment 5: Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.
Response: The symbol "±" has been replaced with descriptive phrasing (standard deviation, SD).

Results Reviewer Comment 1: Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.
Response: Percentages of Trigonelline in all samples have been added to Table 4. However, direct comparisons with literature values are not feasible because this study focuses on developing a green-sensitive method for trace analysis rather than optimizing an extraction method for high yields. Our approach utilized intact, uncrushed fenugreek seeds, whereas previous studies employed powdered seeds, which likely resulted in significantly lower TG %. Despite this difference, our method provides a rapid and reliable method to detect trace amounts of Trigonelline in fenugreek samples.

Discussion Reviewer Comment 1: The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Response: The first paragraph of the discussion has been revised to eliminate repetitive content. It now focuses on the study's findings and how they address the research gap.

Reviewer Comment 2: Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.
Response: The findings for TG content, expressed in percentages, have been compared with values from the literature to provide a clearer context and emphasize the significance of our results.
Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain the current title because the study has already been published. Changing the title could cause confusion and inconsistency in citations and references to the work. However, we have ensured that the terms "green" and "UPLC-MS/MS" are included in the "Keywords" section to emphasize the innovative and advantageous aspects of the study.

Abstract Reviewer Comment 1: Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Response: The abstract has been revised to better articulate the research gap and the limitations of traditional methods, including their inefficiency, reliance on high solvent volumes, and lack of sensitivity.

Reviewer Comment 2: Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
Response: The brackets around "(TG)" have been removed.

Reviewer Comment 3: Include the units for extract yield and TG concentration in the results section for clarity.
Response: Units for extract yield (mg/100mg) and TG concentration (ppb) have been added to the results section of the abstract.

Formatting Reviewer Comment 4: Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Response: The manuscript has been reviewed, and we have ensured that "UPLC-MS/MS" is consistently formatted throughout.

Reviewer Comment 5: Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.
Response: The symbol "±" has been replaced with descriptive phrasing (standard deviation, SD).

Results Reviewer Comment 1: Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.
Response: Percentages of Trigonelline in all samples have been added to Table 4. However, direct comparisons with literature values are not feasible because this study focuses on developing a green-sensitive method for trace analysis rather than optimizing an extraction method for high yields. Our approach utilized intact, uncrushed fenugreek seeds, whereas previous studies employed powdered seeds, which likely resulted in significantly lower TG %. Despite this difference, our method provides a rapid and reliable method to detect trace amounts of Trigonelline in fenugreek samples.

Discussion Reviewer Comment 1: The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Response: The first paragraph of the discussion has been revised to eliminate repetitive content. It now focuses on the study's findings and how they address the research gap.

Reviewer Comment 2: Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.
Response: The findings for TG content, expressed in percentages, have been compared with values from the literature to provide a clearer context and emphasize the significance of our results.
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Author Response 04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

04 Dec 2024

Author Response

Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain ... Continue reading Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain the current title because the study has already been published. Changing the title could cause confusion and inconsistency in citations and references to the work. However, we have ensured that the terms "green" and "UPLC-MS/MS" are included in the "Keywords" section to emphasize the innovative and advantageous aspects of the study.

Abstract Reviewer Comment 1: Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Response: The abstract has been revised to better articulate the research gap and the limitations of traditional methods, including their inefficiency, reliance on high solvent volumes, and lack of sensitivity.

Reviewer Comment 2: Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
Response: The brackets around "(TG)" have been removed.

Reviewer Comment 3: Include the units for extract yield and TG concentration in the results section for clarity.
Response: Units for extract yield (mg/100mg) and TG concentration (ppb) have been added to the results section of the abstract.

Formatting Reviewer Comment 4: Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Response: The manuscript has been reviewed, and we have ensured that "UPLC-MS/MS" is consistently formatted throughout.

Reviewer Comment 5: Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.
Response: The symbol "±" has been replaced with descriptive phrasing (standard deviation, SD).

Results Reviewer Comment 1: Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.
Response: Percentages of Trigonelline in all samples have been added to Table 4. However, direct comparisons with literature values are not feasible because this study focuses on developing a green-sensitive method for trace analysis rather than optimizing an extraction method for high yields. Our approach utilized intact, uncrushed fenugreek seeds, whereas previous studies employed powdered seeds, which likely resulted in significantly lower TG %. Despite this difference, our method provides a rapid and reliable method to detect trace amounts of Trigonelline in fenugreek samples.

Discussion Reviewer Comment 1: The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Response: The first paragraph of the discussion has been revised to eliminate repetitive content. It now focuses on the study's findings and how they address the research gap.

Reviewer Comment 2: Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.
Response: The findings for TG content, expressed in percentages, have been compared with values from the literature to provide a clearer context and emphasize the significance of our results.
Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain the current title because the study has already been published. Changing the title could cause confusion and inconsistency in citations and references to the work. However, we have ensured that the terms "green" and "UPLC-MS/MS" are included in the "Keywords" section to emphasize the innovative and advantageous aspects of the study.

Abstract Reviewer Comment 1: Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Response: The abstract has been revised to better articulate the research gap and the limitations of traditional methods, including their inefficiency, reliance on high solvent volumes, and lack of sensitivity.

Reviewer Comment 2: Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
Response: The brackets around "(TG)" have been removed.

Reviewer Comment 3: Include the units for extract yield and TG concentration in the results section for clarity.
Response: Units for extract yield (mg/100mg) and TG concentration (ppb) have been added to the results section of the abstract.

Formatting Reviewer Comment 4: Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Response: The manuscript has been reviewed, and we have ensured that "UPLC-MS/MS" is consistently formatted throughout.

Reviewer Comment 5: Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.
Response: The symbol "±" has been replaced with descriptive phrasing (standard deviation, SD).

Results Reviewer Comment 1: Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.
Response: Percentages of Trigonelline in all samples have been added to Table 4. However, direct comparisons with literature values are not feasible because this study focuses on developing a green-sensitive method for trace analysis rather than optimizing an extraction method for high yields. Our approach utilized intact, uncrushed fenugreek seeds, whereas previous studies employed powdered seeds, which likely resulted in significantly lower TG %. Despite this difference, our method provides a rapid and reliable method to detect trace amounts of Trigonelline in fenugreek samples.

Discussion Reviewer Comment 1: The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Response: The first paragraph of the discussion has been revised to eliminate repetitive content. It now focuses on the study's findings and how they address the research gap.

Reviewer Comment 2: Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.
Response: The findings for TG content, expressed in percentages, have been compared with values from the literature to provide a clearer context and emphasize the significance of our results.
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Abdulrahman Koshak, King Abdulaziz University, Jeddah, Saudi Arabia
Abdul Sadiq, University of Malakand, Chakdara, Pakistan

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31 Dec 2024 | for Version 3

Abdul Sadiq, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan

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Approved

I approve this manuscript

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Pharmaceutical Sciences

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23 Dec 2024 | for Version 3

Abdulrahman Koshak, King Abdulaziz University, Jeddah, Saudi Arabia

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Approved

I confirm that the authors have addressed and resolved all comments on the manuscript, and I approve it for indexing.

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12 Dec 2024 | for Version 2

Abdulrahman Koshak, King Abdulaziz University, Jeddah, Saudi Arabia

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Approved With Reservations

The manuscript is much clearer now; however, there are still two minor comments to address:

Under the section Method Validation for Green UPLC-MS/MS Analysis, what do you mean by "UPLCMSMS-MD"? Is it the same as UPLC-MS/MS? Please clarify.
It is evident that the percentages of TG differ significantly from those reported in the literature. You attributed this to the use of unprocessed seeds, which is a valuable observation. I suggest emphasizing the importance of seed crushing in releasing the active constituents of the plant. Additionally, you might want to highlight that certain traditional preparation methods, such as making Trigonella tea from uncrushed seeds, could result in a limited extraction of TG.

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Pharmacognosy and natural products

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18 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

Response to the reviewer comment No. 1:
This typo error and we modified it in the manuscript into UPLC-MS/MS

Response to the reviewer comment No. 2
The higher TG concentration observed in these previous studies can be explained by the preparation and crushing of the fenugreek sample into powder, which facilitates more efficient solvent penetration and extraction of bioactive compounds like TG. In contrast, the current study used intact seed to mimic traditional preparation methods where crushing and grinding may not be performed. This highlights the potential limitations of traditional methods such as making fenugreek tea, which result in lower TG extraction yield. Further, the findings emphasise the critical role of the seed preparation in maximising the release of active constituents and underscore the importance of considering these factors when comparing TG concentrations across different studies and extraction methods.

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06 Dec 2024 | for Version 2

Abdul Sadiq, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan

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Approved

I approve the manuscript in the current form

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26 Nov 2024 | for Version 1

Abdul Sadiq, University of Malakand, Chakdara, Khyber Pakhtunkhwa, Pakistan

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Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pharmaceutical Sciences

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04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

Reviewer Comment 1: Phytochemicals usually refer to biologically active compounds produced by plants, which typically include non-nutritive substances such as alkaloids, flavonoids, phenolic acids, tannins, terpenes, and saponins. If proteins or amino acids are mentioned in the context of plant composition, it is more accurate to separate them from the term "phytochemicals." A clearer phrasing for the description of fenugreek seeds might be: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."
Response: We agree with the reviewer’s comment regarding the definition of phytochemicals. The sentence in the abstract has been revised to separate proteins and amino acids from the phytochemical category for accuracy. The updated sentence now reads: "Fenugreek seeds are rich in proteins, lipids, and essential nutrients, and they also contain various phytochemicals, including flavonoids, steroidal saponins, coumarin, and alkaloids such as trigonelline."

Reviewer Comment 2: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North African areas, India, and other countries." The phrase "and other countries" is unnecessary after "including." Consider revising to avoid redundancy.
Response: We appreciate the reviewer’s suggestion to avoid redundancy. The sentence has been revised to remove "and other countries" for conciseness. It now reads: "Fenugreek is cultivated in several countries worldwide, including the Mediterranean region, North Africa, and India."

Reviewer Comment 3: "The USE protocol was selected due to the range of advantages USE presents, including high extraction efficiency and the use of the least possible sample amount, solvent volume, and extraction time." The phrase "the range of advantages USE presents" could be simplified to "the numerous advantages of USE."
Response: We agree with the reviewer’s suggestion to simplify this sentence. The revised sentence now reads: "The USE protocol was selected due to the numerous advantages of USE."

Reviewer Comment 4: "The method avoids the need for an extra step of seed crushing and sieving via the use of an ultrasonic dismembrator with small amounts of sample (10 mg) and low solvent volumes." Consider breaking this into two sentences for clarity, as the crushing and sieving highlight the simplicity of the method, while the use of small sample amounts and solvent volumes emphasizes cost-efficiency and eco-friendliness.
Response: We appreciate this suggestion to enhance clarity. The sentence has been split into two, as follows: "The method avoids the need for additional steps, such as seed crushing and sieving, by using an ultrasonic dismembrator. Furthermore, the method employs small sample amounts (10 mg) and low solvent volumes, ensuring cost-efficiency and eco-friendliness."

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22 Nov 2024 | for Version 1

Abdulrahman Koshak, King Abdulaziz University, Jeddah, Saudi Arabia

25 Views Cite this report Responses(1)

Approved With Reservations

Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
In the results section, include the units for extract yield and TG concentration for clarity.

Formatting:

Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.

Results:

Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.

Discussion:

The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pharmacognosy and natural products

Respond to this report

Responses (1)

Author Response

04 Dec 2024

Salma Hago, Faculty of Pharmacy, Department of Pharmacognosy, University of Gezira, Wad Madani, Sudan

Title Reviewer Comment: Include the keywords "green" and "UPLC-MS/MS" to emphasize the innovative and advantageous aspects of the study. Response: While we appreciate this suggestion, we have chosen to retain the current title because the study has already been published. Changing the title could cause confusion and inconsistency in citations and references to the work. However, we have ensured that the terms "green" and "UPLC-MS/MS" are included in the "Keywords" section to emphasize the innovative and advantageous aspects of the study.

Abstract Reviewer Comment 1: Clearly articulate the research gap and the drawbacks of traditional methods for quality analysis of fenugreek.
Response: The abstract has been revised to better articulate the research gap and the limitations of traditional methods, including their inefficiency, reliance on high solvent volumes, and lack of sensitivity.

Reviewer Comment 2: Avoid mentioning (TG) in brackets, as it has already been identified earlier in the text.
Response: The brackets around "(TG)" have been removed.

Reviewer Comment 3: Include the units for extract yield and TG concentration in the results section for clarity.
Response: Units for extract yield (mg/100mg) and TG concentration (ppb) have been added to the results section of the abstract.

Formatting Reviewer Comment 4: Ensure the correct format (UPLC-MS/MS) is consistently used throughout the article.
Response: The manuscript has been reviewed, and we have ensured that "UPLC-MS/MS" is consistently formatted throughout.

Reviewer Comment 5: Avoid using the symbol "±" for reporting Standard Deviation; opt for a more descriptive format instead.
Response: The symbol "±" has been replaced with descriptive phrasing (standard deviation, SD).

Results Reviewer Comment 1: Provide the percentages of Trigonelline found in all samples to facilitate comparisons with values reported in the literature.
Response: Percentages of Trigonelline in all samples have been added to Table 4. However, direct comparisons with literature values are not feasible because this study focuses on developing a green-sensitive method for trace analysis rather than optimizing an extraction method for high yields. Our approach utilized intact, uncrushed fenugreek seeds, whereas previous studies employed powdered seeds, which likely resulted in significantly lower TG %. Despite this difference, our method provides a rapid and reliable method to detect trace amounts of Trigonelline in fenugreek samples.

Discussion Reviewer Comment 1: The first paragraph contains repetitive information regarding the introduction and methodology. Revise this section to focus directly on the findings and the specific research gap addressed by the study.
Response: The first paragraph of the discussion has been revised to eliminate repetitive content. It now focuses on the study's findings and how they address the research gap.

Reviewer Comment 2: Compare your findings for the amount of TG with those in the literature, expressed in percentages, to provide a clearer context and strengthen the relevance of your results.
Response: The findings for TG content, expressed in percentages, have been compared with values from the literature to provide a clearer context and emphasize the significance of our results.

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Competing Interests

No competing interest

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[2] 2. Garg RC: Fenugreek. Nutraceuticals. Elsevier; 2016; pp. 599–617. Publisher Full Text

[3] 3. Zhou J, Chan L, Zhou S: Trigonelline: A Plant Alkaloid with Therapeutic Potential for Diabetes and Central Nervous System Disease. Curr. Med. Chem. 2012; 19: 3523–3531. PubMed Abstract | Publisher Full Text

[4] 4. Faisal Z, Irfan R, Akram N, et al.: The multifaceted potential of fenugreek seeds: From health benefits to food and nanotechnology applications. Food Sci. Nutr. 2024; 12: 2294–2310. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Membrez M, Migliavacca E, Christen S, et al.: Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia. Nat. Metab. 2024; 6: 433–447. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Farid MM, Yang X, Kuboyama T, et al.: Trigonelline recovers memory function in Alzheimer’s disease model mice: evidence of brain penetration and target molecule. Sci. Rep. 2020; 10: 16424. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Liu H, Li Q, Shao J, et al.: Determination of trigonelline in coffee powder and instant coffee by ultrasonic extraction and high performance liquid chromatography. Se pu Chinese J. Chromatogr. 2011; 29: 1103–1106.

[8] 8. Atlabachew M, Abebe A, Alemneh T, et al.: Rapid and simultaneous determination of trigonelline, caffeine, and chlorogenic acid in green coffee bean extract. Food Sci. Nutr. 2021; 9: 5028–5035. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Al-Juhaimi F, Adiamo OQ, Ghafoor K, et al.: Optimization of ultrasonic-assisted extraction of phenolic compounds from fenugreek (Trigonella foenum-graecum L.) seed. CyTA - J. Food. 2015; 14(3): 369–374. Publisher Full Text

[10] 10. Zhai X, Yang M, Zhang J, et al.: Feasibility of Ultrasound-Assisted Extraction for Accelerated Cold Brew Coffee Processing: Characterization and Comparison With Conventional Brewing Methods. Front. Nutr. 2022; 9: 9. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Singh P, Bajpai V, Gond V, et al.: Determination of Bioactive Compounds of Fenugreek (Trigonella foenum-graecum) Seeds Using LC-MS Techniques. Methods Mol. Biol. 2107; 377–393. PubMed Abstract | Publisher Full Text

[12] 12. Wadhwa G, Krishna KV, Taliyan R, et al.: A novel UPLC–MS/MS method for simultaneous quantification of trigonelline, 4-hydroxyisoleucine, and diosgenin from Trigonella foenum-graecum extract: Application to pharmacokinetic study in healthy and type 2 diabetic rats. Biomed. Chromatogr. 2022; 36: e5275. PubMed Abstract | Publisher Full Text

[13] 13. Ahmad R, Alqathama A, Aldholmi M, et al.: Ultrasonic-assisted extraction of fenugreek flavonoids and its geographical-based comparative evaluation using green UHPLC-DAD analysis. Ultrason. Sonochem. 2023; 95: 106382. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Ahmad R, Aldholmi M, Alqathama A, et al.: Green and novel ultrasonic extraction with UHPLC-MSMS analysis of natural sweetener (Glycyrrhizic acid) from Glycyrrhiza glabra; a multifactorial mechanistic evaluation based on statistical analysis. Ultrason. Sonochem. 2021; 77: 105696. PubMed Abstract | Publisher Full Text | Free Full Text

[15] 15. International conference on harmonisation of technical requirement for registration of pharmaceuticals for human use Ich Harmonised Tripartite Guideline Validation of Analytical P Rocedures.1994.

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A Validated Trigonelline-Based Method for the Standardization and Quality Control of Trigonella foenum-graecum L.

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Figure 1. Chemical structures of trigonelline and nicotinic acid.

Methods

Solvents, chemicals, and instruments

Fenugreek samples collection and preparation

Green Ultrasonics Extraction (USE) for TG

Green UPLCMSMS analytical method for TG

Applicability of the USE-UPLCMSMS for trace analysis

Results

Extracts yield

Table 1. Average and individual extract yields for fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B), and H2O (C).

Table 2. Yield/Origin (mg) and total extract yield (mg) in green solvents for fenugreek samples with SD.

Chromatographic separation and MS optimisation

Table 3. LC and MS conditions for the analysis of TG in fenugreek samples.

Figure 2. UPLCMSMS chromatogram of TG standard.

Figure 3. MSMS fragmentation pattern of TG (m/z).

Figure 4. Standard calibration curve of TG.

TG quantification

Table 4. Average and individual TG amounts in fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B) and H2O (C).

Table 5. TG amount/Origin (ppb) and total TG amount (ppb) quantified via UPLCMSMS in the green extracts of fenugreek.

Applicability of USE-UPLCMSMS for trace analysis

Figure 5. Trace analysis of TG in 10 mg of five fenugreek samples.

Discussion

Conclusions

Ethics and consent

Authors’ contributions

Data and software availability

Underlying data

Software availability statement

Acknowledgements

References

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Table 1. Average and individual extract yields for fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B), and H₂O (C).

Table 4. Average and individual TG amounts in fenugreek samples (F1-F15) extracted with ACt (A), EtOH (B) and H₂O (C).