|Year : 2019 | Volume
| Issue : 4 | Page : 262-272
Screening of herbal medicines for potential allopathic antidiabetic adulterants: An analytical study
Girish Pamidimarri1, Jayanthi Mathaiyan1, S Manikandan1, S Rajan1, Gitanjali Batmanabane2
1 Department of Pharmacology, JIPMER, Puducherry, India
2 Department of Pharmacology, JIPMER, Puducherry; Director, All India Institute of Medical Sciences, Bhubaneswar, Odisha, India
|Date of Submission||12-Sep-2019|
|Date of Decision||07-Jul-2020|
|Date of Acceptance||07-Aug-2020|
|Date of Web Publication||14-Jan-2021|
Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry - 605 006
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: There are several reports worldwide on adulteration of herbal medicines (HMs) with allopathic drugs. In India, only a few studies have reported adulteration of HMs with antidiabetics and there are no systematic studies. Aims: To develop a rapid and validated method for detection of allopathic antidiabetic adulterants and to explore the extent of adulteration in HMs sold in South India. Materials and Methods: Standards and solvents were purchased from Sigma-Aldrich. Different brands of antidiabetic HM samples with manufacturing licenses were procured from dispensaries. Spiked drug free psyllium husk as solid and flask seed oil as liquid herbal matrices were used for method development. The spiked matrices with different concentrations were extracted with methanol and subjected to centrifugation. The supernatant was collected and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isocratic elution was carried on a C18 column with 0.1% (v/v) formic acid:methanol (3:7, v/v) as a mobile phase. All drugs were monitored for two ion products in positive electrospray ionization mode using multiple reaction monitoring scans. Results: The retention time was 9 min. Limit of detection is 10 Pictograms (pg) for all analytes except for metformin, which was 370 pg. Recoveries of analytes range from 96% to 117%. Forty different brands of antidiabetic HMs were analyzed. Adulterant peaks were not observed in the mass chromatograms of HMs. Conclusions: A single-run method was developed by LC-MS/MS for the detection of proposed antidiabetics in HMs from licensed manufacturing units and online sold HMs across herbal dispensaries in Puducherry union territory, India. None of the HMs was found to be adulterated with proposed allopathic antidiabetic adulterants.
Keywords: Adulteration, antidiabetic drugs, Indian traditional medicines, liquid chromatography-triple quadrupole detector, mass spectrometry
|How to cite this article:|
Pamidimarri G, Mathaiyan J, Manikandan S, Rajan S, Batmanabane G. Screening of herbal medicines for potential allopathic antidiabetic adulterants: An analytical study. AYU 2019;40:262-72
|How to cite this URL:|
Pamidimarri G, Mathaiyan J, Manikandan S, Rajan S, Batmanabane G. Screening of herbal medicines for potential allopathic antidiabetic adulterants: An analytical study. AYU [serial online] 2019 [cited 2021 Jul 25];40:262-72. Available from: https://www.ayujournal.org/text.asp?2019/40/4/262/307020
| Introduction|| |
Herbal medicines (HMs) have gathered increasing recognition in recent years, as one of the treatment options. Nearly 65% of the rural population in India depends on the HMs and about 40%–50% of people in Germany, 42% in the USA, 48% in Australia and 49% in France are using traditional medicine. HMs originated from natural sources such as medicinal plants, minerals, animal products and their combinations are sometime presumed to be devoid of adverse effects. Various brands of pharmaceutical formulations which include proprietary and classical types of HMs are available for the treatment of diabetes as over-the-counter medicines.
The safety of HMs is questionable. It has been reported that HMs are adulterated with allopathic drugs.,,,, A few reports of HMs reveals the presence of chlorpropamide, glibenclamide, and metformin as allopathic adulterants.,, A study conducted by Bogusz et al. found sildenafil, tadalafil, testosterone and glibenclamide as adulterants in various herbal remedies. A retrospective study was done by Ching et al. in Hong Kong and found registered and banned antidiabetic allopathic adulterants such as glibenclamide, phenformin, metformin, rosiglitazone, gliclazide, glimepiride, nateglinide and repaglinide in herbal antidiabetic products. Unaware consumption of adulterated HMs may lead to unwanted effects. Phenformin and rosiglitazone are banned for the treatment of type-2 diabetes due to lactic acidosis and heart failure.
The development of novel chromatographic methods for the detection of drugs is an ongoing process. Unlike conventional chromatographic techniques, LC-MS detects the analytes based on their ratio of mass to charge (m/z). Hence, there are fewer chances of false positive. Tandem mass spectrometry (tandem means coupling of two mass spectrometers) (MS/MS) uses both parent and product ion m/z for the detection and also avoids the crosstalk between the analytes, unlike in a solely equipped mass spectrometry, where the detection is based only on the precursor ion m/z.
Solid-phase extraction is one of the extraction methods, which involves the use of more resources and later vaporization techniques make the method cumbersome as liquid chromatography-tandem mass spectrometry (LC-MS/MS) needs a pure form of sample to minimize the difficulty in interpretation. Use of non-deuterated compounds as internal standards is reported and has found no variation in method development. Several methods were developed and reported for the extraction and detection of antidiabetic drugs in human plasma and urine in individuals or other combinations of drugs using different techniques.,,,,, LC-MS/MS is one among them. Electrospray ionization (ESI) is one of the soft ionization techniques, which is commonly used for the ionization of small molecules. Very few studies have reported on the detection of antidiabetic drugs in the herbal matrices., These studies had limitations such as the usage of more solvents and relatively consume more run time. As of our knowledge, there are no systematic studies on the adulterations of HMs with antidiabetics in India. Hence, the current study aims to develop a rapid and validated LC-MS/MS method for the detection of potential allopathic antidiabetic adulterants (metformin, glibenclamide, glimepiride, glipizide, gliclazide and rosiglitazone) in herbal matrices and to examine the problem of adulteration of herbal antidiabetic products which are available in the Puducherry union territory (PUT) region and through web stores.
| Materials and Methods|| |
Materials and reagents
The reference standards (rosiglitazone, glimepiride, metformin HCl, glipizide, glibenclamide and gliclazide), internal standard (IS) (clomipramine HCl) and formic acid were obtained from Sigma-Aldrich, Bengaluru, India. LC-MS grade methanol was purchased from Honeywell, India. Milli–Q water was used, from an in-house water purification system Milli-Q, Merck, Darmstadt, Germany.
LC-MS/MS analyses were performed on a Waters e2695 separation module and Waters Acquity TQ detector (Waters, Milford, CT, USA). Nebulizer gas (nitrogen) was generated by the NM32 LA Peak (Peak scientific, Billerica, MA, USA). Argon gas was used as collision gas (INOX, Chennai, TN, INDIA). ESI was operated in the positive and negative mode.
Symmetry C18 reverse-phase column (specifications: 150 mm × 3.9 mm i.d., particle size 5 μm) by Waters was used for the analyses. The mobile phase was composed of Milli-Q water containing 0.1% formic acid as solvent A and 100% of methanol as solvent B. An isocratic elution was done throughout the analyses, which contained 30% solvent A and 70% solvent B. The LC column temperature was set at 30°C and the flow rate was 0.6 ml/min.
Tandem mass spectrometry
ESI was used for ionization. TQD type of tandem mass spectrometry was used and it was performed in multiple reaction monitoring mode, to detect the multiple parents and ion products. The resolution was set at 1 Da. The cone voltage (CV) and collision energy (CE) settings for these drugs ranged from 20 to 42 V and 12–36 eV respectively. Argon gas was used as an inert gas in collision-induced dissociation. A full-scan MS acquisition of the mass range between 100 and 600 was used.
MassLynx V4.1 software was used for data acquisition and processing which was provided by the Waters. IntelliStart, which is a part of it, and was used for the development of methods.
Stock solutions and herbal matrices
Stock solutions (1 mg/ml) of reference standards and IS were prepared by adding in methanol (LC-MS grade) as a solvent. Psyllium husk (manufactured by Organic India Pvt. Ltd., Lucknow, India), which is commonly used as laxative, was taken as a solid matrix. Flaxseed oil (packaged by Loose Square Marketing solutions, Haryana, India), which is commonly used as a weight-loss supplement, was taken as a liquid matrix. Both these matrices were confirmed to be devoid of adulterants (glibenclamide, glimepiride, glipizide, gliclazide, metformin and rosiglitazone) by LC-ESI-MS/MS. An equal amount of IS (400 ng) spiked in all standards.
Preparation of calibration standards in a solid herbal matrix
Calibration standards were prepared by appropriate dilutions of stock solution in methanol. Ten μl of the working standards was used to prepare different concentrations of all the drugs (metformin, glibenclamide, glimepiride, glipizide, gliclazide and rosiglitazone) to yield 100 ng, 200 ng, 400 ng, 800 ng and 1000 ng in 1 g of the solid matrix. All standard solutions were stored at −30°C.
Preparation of calibration standards in a liquid herbal matrix
Calibration standards were prepared by appropriate dilutions of stock solution in methanol. Ten μl of the working standards was used to prepare a different concentration of all the drugs (metformin, glibenclamide, glimepiride, glipizide, gliclazide and rosiglitazone) to yield 100 ng, 200 ng, 400 ng, 800 ng and 1000 ng in 1 ml of liquid matrix. All standard solutions were stored at −30°C.
The 1 g or 1 ml of blank matrices and spiked matrices (with standards and IS) were extracted by dissolving with 10 ml of LC-MS grade methanol in 15 ml tarson tube and then centrifuged using Sorvall Legend XTR centrifuge for 5 min at 3600 RPM. Two ml of the supernatant was collected and again centrifuged by using Mikro 22R zentrifugen for 3 min at 16,000 RPM. Finally, 1 ml of the supernatant was collected and used for LC-ESI-MS/MS analysis. Solid samples such as tablets or capsules were powdered using an agate mortar and pestle and followed the same extraction procedure.
The solutions of pure reference drugs and extracted solutions of spiked solid and liquid herbal matrices were subjected to intraday and interday analysis. From this analysis, the mean retention time and relative standard deviations were calculated. Recoveries in both matrices were calculated by AUC (spiked matrix) divided by AUC (standard) and multiplied by 100. Limit of detection (LOD) and limit of quantification (LOQ) was determined by the standard deviation of low concentration to the slope of the calibration curve signal to noise ratio 3 and 10, respectively.
This is an analytical experimental study on the analysis of HMs for the presence of potential allopathic antidiabetic adulterants. The adulterants selected for the study are metformin, glibenclamide, glimepiride, glipizide, gliclazide, and rosiglitazone and were based on the literature. After obtaining the necessary permission from the Institute scientific advisory committee and Institute ethics committee, the HM samples were procured from licensed dispensaries. All samples were collected in triplicates from various herbal dispensaries in the PUT region of India and through online stores with an intact seal. PUT consists of four districts Puducherry, Karaikal, Mahe and Yanam. The medicines that have a manufacturing license were included for analyses. The solid and liquid herbal matrices were first checked for the absence of proposed antidiabetic drugs using LC-ESI-MS/MS and were considered as blanks. Spiked HM samples with standards and IS were extracted using 10 ml of LS-MS grade methanol. This step was followed by centrifugations, as mentioned in the extraction subheading.
Formic acid was used as one of the mobile phases at 0.1% concentration. It acts as a proton source, that is useful in the ionization of the analytes into [M+H]+ ions, which is also useful for the analysis of analytes in positive ionization mode. It has also an advantage of not being a strong ion-pair agent, unlike trifluoroacetic acid, and it does not suppress MS ionization. The injection volume was 25 μl. and 0.6 ml/min kept as flow rate. All mobile phases were sonicated before use. Isocratic elution was applied using 30% of 0.1% formic acid in Milli-Q H20 (A) and 70% of methanol in Milli-Q H20 (B). This elution method was selected by trial and error method starting from 50% of A and B solvents, 40% of A and 60% of B, 30% of A and 70% of B and 35% of A and 65% of B (data not shown). Monoisotopic mass was considered as an initial mass of all the analytes since all were small molecules.
| Results|| |
A total of forty different brands of antidiabetic HMs were purchased. Samples purchased from each district and through web stores are mentioned in [Table 1]. Out of which, thirty seven were solid and 3 were liquid dosage formulations. The price range of the samples was between 23 and 622 INR, which includes both proprietary (35 samples) and classical (5 samples) HMs. Percentage of recoveries is in the range of 98%–117% and 99%–110% in liquid and solid matrices, respectively [Table 2]. Intraday and interday analyses were done for both spiked solid and liquid matrices in five different concentrations. The percentage of relative standard deviation of the five different calibration standards was under 15% [Table 3]. A difference in the retention time of metformin was observed, which was higher in the solid matrix than in the liquid matrix, but there was no change in the mean runtime. Total runtime was 12 min, which includes 3 min as an extra runtime to avoid carryover effect [Table 4]. LOD and LOQ are given in [Table 2].
|Table 1: Total number of herbal medicines of different brands collected from different districts in Puducherry union territory and through online|
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|Table 3: Intra-day and inter-day analysis of antidiabetics in solid and liquid matrices|
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The mass chromatograms of blank solid and liquid matrices are shown in [Figure 1] and [Figure 2], respectively. The mass chromatograms of the antidiabetic standards are depicted in [Figure 3]. The mass chromatograms of the antidiabetics spiked in a solid and liquid matrix are shown in [Figure 4] and [Figure 5], respectively. The representative mass chromatograms of solid and liquid HM spiked with IS are shown in [Figure 6] and [Figure 7], respectively. Finally, the mass chromatograms of intentionally spiked with antidiabetic standards are depicted in [Figure 8] and [Figure 9], respectively.
|Figure 1: Mass chromatogram of blank solid matrix – Psyllium husk; No peaks of glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide, clomipramine (IS) and metformin|
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|Figure 2: Mass chromatogram of blank liquid matrix – Flax seed oil; No peaks of glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide, clomipramine (IS) and metformin|
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|Figure 3: Mass chromatogram of reference standards (in methanol): Retention time and abundance of glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide, clomipramine (IS) and metformin|
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|Figure 4: Mass chromatogram of the spiked solid matrix: Retention time and abundance of glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide, clomipramine (IS), and metformin|
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|Figure 5: Mass chromatogram of the spiked liquid matrix: Retention time and abundance of glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide, clomipramine (IS), and metformin (from top to bottom)|
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|Figure 6: Mass chromatogram of solid HM sample number 3: Presence of clomipramine (IS) peak only and the absence of proposed adulterants peaks|
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|Figure 7: Mass chromatogram of liquid AHM sample number 11. clomipramine (IS) peak only present and the absence of proposed adulterants peak|
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|Figure 8: Mass chromatogram of intentionally spiked solid HM sample number 24 shows peaks of all antidiabetic analytes|
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|Figure 9: Mass chromatogram of intentionally spiked liquid HM sample number 20 show peaks of all proposed anti-diabetic adulterants|
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The obtained mass chromatograms from the HMs spiked with IS were compared with mass chromatograms of the herbal matrices spiked with proposed antidiabetic adulterants and IS. The absence of the respective peaks in the mass chromatograms of HMs reveals the absence of the adulterants. None of the mass chromatograms of HMs showed adulterant peaks other than IS peak, which confirms that the samples analyzed are devoid of the proposed adulterants. Finally, HMs were analyzed with the intentionally spiked proposed possible adulterants, to overcome false-negative findings that may happen at the time of extraction due to degradation. All proposed adulterant peaks were seen in mass chromatograms of intentionally spiked HMs [Figure 8].
| Discussion|| |
In this study, a total of forty antidiabetic HMs were collected, out of which thirty five were proprietary and the remaining 5 were classical preparations. The details of 5 classical preparations from which they were formulated are as follows:
- One preparation is of siddha formulation - Keezhanelli tablets, formulated with reference to Vaidyaratna Surukkam [Table 5].,
- Four preparation were of Ayurvedic formulations - Nisha Amalaki churnam, and Nisha Amalaki powder are formulated with reference to Ashtangahridaya. Panchabana Rasa formulated with reference to Yogaratnakara. Abhraka Bhasma formulated with reference to Rasarantna samuchaya [Table 5].,,,
Among purchased proprietary medicines there were total 28 Ayurvedic proprietary medicines, 6 Siddha proprietary medicines and one herbal proprietary anti-diabetic medicine were monitored. The method for monitoring was developed as follows,
Chromatographic parameters such as mobile phase consists of 30% of (0.1% formic acid in milli Q water) as solvent A and 70% of 100% methanol as solvent B was eluted through reverse phase column (150*3.9 mm, particle size 5μm) at 300c, flow rate0.6m/min. Common method of sample (solid and liquid) preparation was used as. 1 mg/ml stock solution in 100% methanol. For the extraction method, 1 gm/ml sample in 10 ml methanol was centrifuged at 3600 rpm for 5 mins and 16000 rpm for 3 mins. Internal standard (clomipramine) were spiked in herbal solid matrix (psyllium husk) and liquid matrix (flax seed oil). The adulterants choosen were glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide and metformin. The method was validated by percentage recovery, LOD, LOQ on the basis of AUC of spiked IS matrix and sample.
For mass spectrometry, initially all the analytic ions in ESI positive and negative modes and considered the positive ESI mode signals, as these signal intensities for all the spikes were higher than those in negative ion mode. A difference of one proton mass between the initial monoisotopic mass and the precursor ion mass suggests the occurrence of protonation [Table 4]. The protonated form of each analyte including IS was used as the precursor to obtain product ion spectra at third quadrupole. All parameters were optimized with the use of MassLynx software like CV and CE. The procedure complies with ICH harmonized tripartite guideline for the validation of analytical procedures. Clomipramine HCl was used as IS as it is not a part of antidiabetic action and therefore cannot be used as antidiabetic adulterants in HMs. All the analytes for two transitions (data not shown) were scanned and considered the most abundant transition for monitoring. The respective m/z ratios of precursor and product ions of metformin (130.2 > 60.2), glibenclamide (494.1 > 369.1), glimepiride (491.1 > 352.1), glipizide (4461.1 > 321.1), rosiglitazone (358.1 > 135.2) and clomipramine (315.1 > 86.20) were considered for monitoring. The observed m/z ratio of product ions in the study was similar to the studies reported in the literature.,,,,, The respective details are shown in [Table 6]. However, for gliclazide, the observed transition in the present study was different (324.1 > 91.2 vs. 324.1–127.25) from two other reported studies.,
The mass chromatograms of HM samples spiked with IS were compared with the mass chromatograms of herbal matrices spiked with antidiabetic standards and IS. Mass chromatograms of HM sample 3 and 11 showed the presence of only IS peak [Figure 6] and [Figure 7]. The absence of the respective allopathic antidiabetic peak in the HM sample chromatograms reveals the absence of potential adulterants in the analyzed HM samples. Finally, intentionally spiked HM samples were also analyzed because surrogate matrices were used in the method development process and/or to avoid a chance of unidentified due to degradation of the spiked analytes through the extraction procedure. Mass chromatograms of intentionally spiked solid and liquid HM samples showed all the antidiabetic standard peaks [Figure 8] and [Figure 9].
A study by Bogusz et al. used LC–ESI–MS-MS for the detection of antidiabetic drug adulterants such as chlorpropamide, glibenclamide, gliclazide, glimepiride, glipizide, pioglitazone, tolazamide, and tolbutamide in the herbal remedies and found glibenclamide as an adulterant. A retrospective study by Ching et al. on the analysis of the 29 illicit herbal antidiabetic products revealed the presence of eight registered or banned oral antidiabetic adulterants, namely glibenclamide, phenformin, metformin, rosiglitazone, gliclazide, glimepiride, nateglinide and repaglinide as adulterants. A case report by Wood et al., a diabetic patient on a visit to his native in India for approximately after 1 year and on returning to the clinic in the U.K., found to be excellent glycemic control because of three different HMs used. On the analysis of blood and HM, samples using high-performance liquid chromatography-ultraviolet (HPLC-UV) were found to contain chlorpropamide as an adulterant. Another study by Padinjakara et al., patients who had purchased HMs from India, on the chemical analysis found to contain glibenclamide as an adulterant. A case report by Kumar et al. found metformin as an adulterant in the HM by HPLC-UV/IR spectroscopy, which was taken from the local herbal medicinal practitioner. Conversely, in the study, none of the anti-diabetic adulterants in proprietary as well as classical HMs were found which are collected from the local HM dispensaries and through web stores. It might be due to the strict inclusion criteria, i.e., intact samples with manufacturing licenses. The study, thus, indicates the products marketed by licensed manufacturers had no problem of allopathic adulterants and can serve as authentic sources for use.
| Conclusions|| |
A single-run LC-ESI-MS/MS method was developed for the detection of potential allopathic anti-diabetics adulterants in herbal solid and liquid matrices. The method was validated by checking recovery, LOD and LOQ. The developed method has a relatively easy extraction process, quick run time and sensitive detection of adulterants. All the samples of herbal antidiabetic medicine were tested for the presence of adulterants glibenclamide, glimepiride, glipizide, rosiglitazone, gliclazide and metformin. No adulteration was found in the analyzed samples with the proposed adulterants. The developed method can be useful for routine screening of HMs for proposed anti-diabetic adulterants.
Financial support and sponsorship
This study was financially supported by JIPMER intra mural fund.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sen S, Chakraborty R. Toward the integration and advancement of herbal medicine: A focus on traditional Indian medicine. Bot Targets Ther 2015;5:33-44.
Devi MV. 5 – Quality control and assurance of Indian medicines. Health Adm 2007;20:21-5.
Wood DM, Athwal S, Panahloo A. The advantages and disadvantages of a 'herbal' medicine in a patient with diabetes mellitus: A case report. Diabet Med 2004;21:625-7.
Kulambil Padinjakara RN, Ashawesh K, Butt S, Nair R, Patel V. Herbal remedy for diabetes: Two case reports. Exp Clin Endocrinol Diabetes 2009;117:3-5.
Kumar M, Mandal V, Hemalatha S. Detection of metformin hydrochloride in a traditionally used Indian herbal drug for antidiabetic: A case report. Int J Pharma Biosci 2011;2:307-13.
Bogusz MJ, Hassan H, Al-Enazi E, Ibrahim Z, Al-Tufail M. Application of LC-ESI-MS-MS for detection of synthetic adulterants in herbal remedies. J Pharm Biomed Anal 2006;41:554-64.
Ching CK, Lam YH, Chan AY, Mak TW. Adulteration of herbal antidiabetic products with undeclared pharmaceuticals: A case series in Hong Kong. Br J Clin Pharmacol 2012;73:795-800.
Kruve A, Rebane R, Kipper K, Oldekop ML, Evard H, Herodes K, et al
. Tutorial review on validation of liquid chromatography-mass spectrometry methods: Part I. Anal Chim Acta 2015;870:29-44.
Chen L, Zhou Z, Shen M, Ma A. Simultaneous determination and pharmacokinetic study of metformin and rosiglitazone in human plasma by HPLC-ESI-MS. J Chromatogr Sci 2011;49:94-100.
Şenkardeş S, Özaydın T, Uğurlu T, Küçükgüzel ŞG. Development and validation of a reversed-phase HPLC method for the determination of lisinopril and gliclazide in pharmaceuticals. Marmara Pharm J 2017;21:338-44.
Ezzeldin E, Abo-Talib NF, Tammam MH, Shahat AA. Development and validation of LC/MS/MS method for the simultaneous determination of montelukast, gliclazide, and nifedipine and its application to a pharmacokinetic study. Chem Cent J 2014;8:17.
Qiu X, Zheng SL, Wang Y, Wang R, Ye L. A rapid and simple UPLC-MS-MS method for determination of glipizide in human plasma and its application to bioequivalence study. J Chromatogr Sci 2015;53:85-9.
Zhou Z, Zhang J, Zhang W, Bai Y, Liu H. Rapid screening for synthetic antidiabetic drug adulteration in herbal dietary supplements using direct analysis in real time mass spectrometry. Analyst 2011;136:2613-8.
Ding CG, Zhou Z, Ge QH, Zhi XJ, Ma LL. Simultaneous determination of metformin and glipizide in human plasma by liquid chromatography- tandem mass spectrometry. Biomed Chromatogr 2007;21:132-8.
Du Y, Li Q, Wu C, Zhang Y. Rapid screening and quantitative detection of 11 illegally added antidiabetics in health care products by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry. Se Pu 2015;33:371-6.
Goyal A, Sharma V, Upadhyay N, Gill S, Sihag M. Flax and flaxseed oil: An ancient medicine & modern functional food. J Food Sci Technol 2014;51:1633-53.
Yu H, Straubinger RM, Cao J, Wang H, Qu J. Ultra-sensitive quantification of paclitaxel using selective solid-phase extraction in conjunction with reversed-phase capillary liquid chromatography/tandem mass spectrometry. J Chromatogr A 2008;1210:160-7.
Senko MW, Beu SC, McLaffertycor FW. Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J Am Soc Mass Spectrom 1995;6:229-33.
SHASTRI VSL, SHASTRI BSB, Prakashan C, Gupta A. YOGARATNAKARA. Varanasi: Chaukhambha Prakashan; 2017
Tripathi DB. Ashtanga Hridayam. Chaukhambha; 2005. 1379 p.
ICH, Q2(R1). In: International Conference on Harmonisation. Geneva: IFPMA; 2005.
Polagani SR, Pilli NR, Gajula R, Gandu V. Simultaneous determination of atorvastatin, metformin and glimepiride in human plasma by LC-MS/MS and its application to a human pharmacokinetic study. J Pharm Anal 2013;3:9-19.
Ho EN, Yiu KC, Wan TS, Stewart BD, Watkins KL. Detection of anti-diabetics in equine plasma and urine by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2004;811:65-73.
Zhang X, Wang X, Vernikovskaya DI, Fokina VM, Nanovskaya TN, Hankins GD, et al
. Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS. Biomed Chromatogr 2015;29:560-9.
Balsevich G, Namendorf C, Gerlach T, Uhr M, Schmidt MV. The bio-distribution of the antidepressant clomipramine is modulated by chronic stress in mice: Effects on behavior. Front Behav Neurosci 2015;8:445 Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285108/
. [Last accessed on 2018 Dec 12].
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]