Validated UPLC/Q-TOF-MS Method for Simultaneous Determination of Metformin, Glimepiride and Pioglitazone in Human Plasma and its Application to Pharmacokinetic Study

 

Hamid Khan1*, Mushir Ali2, Alka Ahuja2, Javed Ali2

1JK College of Pharmacy, Bilaspur, CG, India-495001

2Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India-110062

*Corresponding Author E-mail: khanhamid770@gmail.com

 

ABSTRACT:

In the presented work the ultra-performance liquid chromatographic/quadrupole time-of-flight mass spectrometric (UPLC/Q-TOF-MS) method has been developed for simultaneous determination of metformin, glimepiride and pioglitazone in human plasma. For identification of drugs, the Q-TOF mass spectrometer was operated in positive ionization mode and quantification was done using the MS/MS transitions at m/z 130.0 to 71.0 for metformin, 491.00 to 352.00 for glimepiride and 357.00 to 134.00 for pioglitazone. The chromatographic separation was achieved on Acquity UPLCTM BEH C18 (100.0 × 2.1 mm, 1.7µm) column using isocratic mobile phase consisting of acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.20 mL/min. The elution of metformin, glimepiride and pioglitazone was occurred at 0.50, 1.40 and 1.22 min, respectively. The calibration curves were linear over the concentration range of 1-1000 ng/mL for all the drugs. The developed method was validated according to ICH guidelines. The method was applied for pharmacokinetic study of drugs in FDC tablets in human plasma.

 

KEY WORDS: UPLC/Q-TOF-MS, Metformin, Glimepiride, Pioglitazone, FDC tablets, Pharmacokinetic Study.

 

 


INTRODUCTION:

The UHPLC/Q-TOF-MS technique is becoming very popular in the identification and quantification of synthetic compounds. The use of conventional techniques, such as HPLC-UV, HPLC-MS and NMR, cannot address high throughput analytical needs due to relatively less sensitivity, high sample purity requirement, necessity of operator expertise and the use of costly solvents. The Q-TOF mass spectrometry gives the accurate mass and reliable fragmentation of synthetic compounds [1-4].

 

Metformin hydrochloride (MET) (N,N-dimethylimidodicarbonimidic diamide hydrochloride) is an orally administered biguanide widely used in the treatment of type 2 (non-insulin dependent) diabetes mellitus. It is antihyperglycemic drug that lowers glucose by reducing hepatic glucose production and gluconeogenesis and by enhancing peripheral glucose uptake [5]. Glimepiride (GLIM) (1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline). As with the other sulphonylureas, glimepiride appears to lower blood glucose levels by stimulating insulin release from the pancreas [6]. Pioglitazone hydrochloride (PIO) [(±)-5-[[4-[2-(5-ethyl-2-pyridinyl) ethoxy] phenyl] methyl]-2,4-] thiazolidine-dione monohydrochloride is an oral anti-hyperglycemic agent which acts primarily by decreasing insulin resistance in the periphery and liver resulting in increased insulin dependent glucose disposal and decreased hepatic glucose output. It is used in the treatment of type-II diabetes mellitus [7]. Fixed dose combination (FDC) tablets containing 500 mg of metformin hydrochloride, 2 mg of glimepiride and 15 mg of pioglitazone hydrochloride has been approved for the treatment of diabeties and widely available in the Indian market.  The literature survey revealed that few analytical methods have been reported for determination of metformin as an individual drug in biological fluids such as LC-MS [8-10]. Determination of glimepiride as an individual drug in biological fluids has been reported by LC-MS [11, 12]. Simultaneous determination of metformin, glimepiride and pioglitazone in formulations and/or biological fluids has been reported by HPLC [13, 14] and by LC-MS-MS [15]. However a UPLC-MS method was developed for simultaneous determination of all the three drugs in tablets but the developed method was found very complicated due to the use of internal standard along with main analytes and also gradient elution for chromatographic separation [16]. Comparatively the method which does not involve use of internal standard minimizes interference from the sample matrix and isocratic elution makes the method easy and fast. Hence in the presented work UPLC/Q-TOF-MS method is developed and validated for identification and quantification of metformin, glimepiride and pioglitazone in human plasma using isocratic elution without use of internal standard.

 

EXPERIMENTAL:

Chemicals and Reagents:

Metformin hydrochloride (C4H11N5.HCl, Molecular weight 165.62, and purity 99.98%), Glimepiride (C24H34N4O5S, Molecular weight 490.62, and purity 99.95%),  and pioglitazone hydrochloride (C19H21ClN2O3S, Molecular weight 392.90, and purity 99.97%) were kindly supplied as gift sample by Systopic Pharmaceuticals Ltd. (New Delhi, India). Tablets (TRIEXER, Cipla) were obtained commercially with labeled amounts of 500 mg of metformin hydrochloride, 2 mg of glimepiride and 15 mg of pioglitazone hydrochloride. LC-MS grade water; acetonitrile, methanol, and ammonium acetate were purchased from Fluka analytical, Sigma-Aldrich Corporation, St. Louis, MO, USA. All other reagents used were of LC-MS grade.

 

Q-TOF-MS and UPLC Conditions:

Mass spectrometry was performed on a Waters Synapt Q-TOF Premier (Micromass MS Technologies, Manchester, UK) mass spectrometer. The Q-TOF mass spectrometer was operated in positive ionization mode and quantification was done using the MS/MS transitions at m/z 130.0 to 71.0 for metformin, 491.00 to 352.00 for glimepiride and 357.00 to 134.00 for pioglitazone. UPLC was performed with Waters Acquity UPLC system (Waters Corporation, MA, USA) equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector.

 

Preparation of Standard Solutions:

Each of metformin, glimepiride and pioglitazone were weighed accurately and transfer to 50 mL volumetric flasks separately. The powders were then dissolved with approximately 25 mL of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. The solutions were further diluted with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound.

 

Preparation of Sample Solutions:

500 μL of plasma sample was transferred to 10 mL glass tube. To this 5 mL of extraction solvent (diethyl ether: dichloromethane 70:30, v/v) was added. The sample was mixed by vortexer for 5 min. The organic layer was transferred to another glass tube. The solid residue was evaporated to dryness using evaporator at 40 ºC under a stream of nitrogen. The dried extract was reconstituted in 200 μL of diluent (methanol: water, 50:50, v/v). This solution was filtered through 0.45 μm nylon membrane filter to remove all the particulate materials. 20 μL aliquot was injected in to UPLC system.

 

VALIDATION OF THE METHOD:

The developed method was validated according to ICH validation guidelines [17]. The validation parameters addressed were linearity and range, limit of detection and quantitation, precision, accuracy, and specificity.

 

Linearity, Range, LOD and LOQ:

Different standard concentrations each of metformin hydrochloride, glimepiride and pioglitazone hydrochloride in the range of 1-1000 ng/mL (1, 10, 50, 100, 200, 500, and 1000 ng/mL) was spiked to 100 μL of blank human plasma separately in methanol: water (50:50, v/v). Similarly the low, medium and high concentration QC samples containing 100, 200 and 400 ng/mL for each drug were prepared independently using the same procedure. The solutions were filtered through 0.20 μm nylon syringe filter and injected in to the UPLC/QTOF-MS system for analysis. Average peak area at each concentration level was subjected to linear regression analysis with the least squares method. Linearity was described by slope, intercept and correlation coefficient obtained from regression equations.

 

ACCURACY AND PRECISION:

Intraday and interday accuracy and precision was evaluated by analyzing low, medium and high concentration QC samples containing 100, 200 and 400 ng/mL of each drug concentration (n=6) on three consecutive days. The mean of percentage recoveries and the RSD (%) was calculated.

 

Specificity:

Specificity is the ability of the method to measure the analyte response in the presence of sample matrix. The specificity of the method was examined by analyzing blank plasma extract. The chromatogram of drug free plasma was compared with the chromatograms obtained from plasma spiked with both analytes.

 

Stability of samples:

Sample stability was tested by analyzing QC samples containing 100 ng/mL of each drug after short-term (6 h) storage at 25 ºC, 12 h storage in an auto sampler at 25 ºC, after three freeze-thaw (-20 ºC) cycles, and after long-term (15 days) storage at -20 ºC. The results were compared with those QC samples freshly prepared and RSD (%) was calculated.

 

Pharmacokinetic Study:

The method was applied for pharmacokinetic study to determine the plasma concentrations of metformin, glimepiride and piogliatazone from a clinical trial in which 3 healthy male volunteers received a FDC tablet (TRIEXER) containing 500 mg metformin hydrochloride, 2 mg glimepiride and 15 mg pioglitazone hydrochloride. Blood samples were collected before and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h post-dosing. Plasma was separated by centrifugation of the heparin zed samples at 2000 × g for 10 min and was stored at -20 ºC until analysis.

 

RESULTS AND DISCUSSION:

All the compounds have strong responses in the positive ionization mode. Therefore, the positive ions, [M+H]+ at m/z 130.00 for metformin hydrochloride, m/z 491.00 for glimepiride and m/z 357.80 for piogliazone hydrochloride were selected as the precursor ions. Under the selected MS/MS conditions the precursor ions were fragmented to major product ions at m/z 130.0 to 71.0 for metformin hydrochloride, 491.00 to 352.00 for glimepiride and 357.00 to 134.00 for pioglitazone hydrochloride, as shown in Figure 1, Figure 2 and Figure 3, respectively. The fragmentation pattern of the compounds and obtained mass spectra in this study was found more authentic and accurate compared to those reported in previously published method [8-12, 16, 17]. Quantification was done by taking the major product ions. The various parameters for Q-TOF-MS and UPLC conditions are presented in Table 1.

 

 

Figure 1. TOF-MS/MS spectra of Metformin hydrochloride

 

Figure 2. TOF-MS/MS spectra of Glimepiride

 

Figure 3. TOF-MS/MS spectra of Pioglitazone hydrochloride

 

Table 1.Various Parameters for Q-TOF-MS and UPLC Conditions

Q-TOF-MS Conditions

UPLC Conditions

Capillary voltage

3.0 kV

Chromatography

Waters Acquity UPLC system

Sampling cone voltage

40 V

Column

Acquity UPLC BEH C18

Extraction cone voltage

4 V

Column dimension

100.0 × 2.1 mm, 1.7µm

Source temperature

80ºC

Mobile phase

Acetonitrile–2 mM ammonium acetate (50:50, v/v)

Cone gas flow

50 L/h

Mobile phase flow rate

0.20 mL/min

Source gas flow

0.50 mL/min

Elution mode

Isocratic

Collision gas (Argon)

2.5×10-4 mbar

Total run time

3 min

Collision energy

12 V

System pressure

2450 to 2500 psi


Optimization of UPLC Conditions:

The isocratic mobile phase containing acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.20 mL/min provide peaks with short retention times. The retention time was found to be 0.50 min for metformin, 1.40 for glimepiride and 2.22 min for pioglitazone with the total chromatographic run time of 3.00 min for each compound. UPLC-TOF-MS/MS chromatogram obtained from mixed standards (1ng/mL each) of all the drugs is shown in Figure 4.

 

Figure 4. UPLC-TOF-MS/MS chromatogram obtained from mixed standards (1ng/mL each) of metformin hydrochloride (Rt  0.50 min),  glimepiride (Rt  1.40 min), and pioglitazone hydrochloride (Rt  2.22 min)

 

VALIDATION OF THE METHOD:

The results of linearity, LOD and LOQ are presented in Table 2. The obtained results indicated that higher sensitivity of the method. The RSD less than 2% were obtained for all the compounds by evaluation of intraday, interday, and different analysts precision suggested an acceptable precision of the method. The results of accuracy and precision are presented in Table 3.

 

Table 2. Results Obtained from Linearity, LOD, and LOQ

Parameters

MET

GLIM

PIO

Linear range (ng/mL)

1-1000

1-1000

1-1000

Correlation coefficienta

0.9997

0.9997

0.9998

LOD (ng/mL)

0.1

0.01

0.01

LOQ (ng/mL)

1

1

1

aMean of six replicates (n = 6).

 

Table 3. Results Obtained from Recovery Studies and Precision

Conc. Added

 (ng/mL)

Conc. Found

 (ng/mL)

Recovery

(%)a

RSD (%)

Intraday

Interday

MET

250

500

 

249.94

500.10

 

99.97 100.02

 

0.78

0.95

 

1.45

 1.75

GLIM

100

200

 

99.92

199.85

 

99.92

99.92

 

1.55

1.12

 

1.45

1.28

PIO

150

300

 

150.10

299.95

 

100.06

99.98

 

1.55

1.34

 

1.45

1.28

aMean of six replicates (n = 6)

Stability of samples:

The stability of drugs in human plasma under various storage conditions and time period are presented in Table 4. The results indicated that no significant change in the concentration of drugs over the period of 12 h at room temperature which was covered the entire chromatographic procedure. There were no significant differences in the concentration of drugs when the samples were subjected to three freeze-thaw (-20 ºC) cycles and after long-term (15 days) storage at -20 ºC (p>0.05, ANOVA).

 

Table 4. Results Obtained from Stability Studies

Storage conditions

Analyte

Conc. Added (ng/mL)

Conc. Found

 (ng/mL)

RSD

 (%)

Storage for 6 h at 25 ºC

MET

GLIM

PIO

500

200

150

499.52

199.12

150.10

1.45

1.34

1.57

Three freeze-thaw  (-20 ºC) cycles

MET

GLIM

PIO

500

200

150

500.15

199.75

149.97

1.54

1.75

1.45

  Storage for 15 days at -20 ºC

MET

GLIM

PIO

500

200

150

500.17

199.15

150.05

1.85

1.94

1.25

 

 

Pharmacokinetic Study:

The method was applied to pharmacokinetic study in human plasma. The results of pharmacokinetic parameters obtained from mean plasma concentration time curve after administration of single FDC tablet containing 500 mg metformin hydrochloride, 2 mg glimepiride and 15 mg pioglitazone hydrochloride are presented in Table 5. The results obtained from pharmacokinetic parameters were not significantly different from the reported methods of each drug [8-12, 15].

 

Table 5: Results Obtained from Pharmacokinetic Studies

Pharmacokinetic Parameter

MET

GLIM

PIO

Tmax (h)

1.50± 0.30

3 ± 0.50

1.5 ± 0.25

Cmax (ng/mL)

950± 100

5065 ± 110

6065 ± 150

AUC (hr.ng/mL)

3550± 550

15150 ± 150

55110 ± 100

T1/2

4.5± 0.50

7.0 ± 2.0

5.0 ± 1.0

Data are mean ± S.D., n = 3

 

CONCLUSION:

This is the first method described for identification and quantification of three anti-diabetic drugs namely metformin, glimepiride and piogliazone in human plasma by application of UPLC/Q-TOF-MS technique. The developed method has shown acceptable precision, accuracy and sensitivity of all the drugs in human plasma samples obtained by pharmacokinetic studies. The use of isocratic chromatographic separation without any internal standard makes it an advantageous analytical method for pharmacokinetic study of all three drugs in  their fixed-dose combination tablets.

 

ACKNOWLEDGMENTS:

The authors are grateful to Systopic Laboratories Ltd., Delhi, India, for providing pure standard samples of metformin, glimepiride and pioglitazone. The authors are also thankful to Dean and In-charge of Instrumentation Facilities, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, New Delhi, India, for providing opportunities to work on UPLC/Q-TOF-MS system.

 

REFERENCES:

1.     Swartz ME. UPLC: An Introduction and Review. Journal of Liquid Chromatography and Related Technology. 2005; 28(1):1253-1263.

2.     Novakova L, Matysova L, Solich P. Advantages of Application of UPLC in Pharmaceutical Analysis. Talanta. 2006; 68(3): 908-918.

3.     Plumb R, Castro-Perez J, Granger J, Beattie I, Joncour K, Wright A. Ultra performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry. Rapid Communication in Mass Spectrometry. 2004; 18(19): 2331-2337.

4.     Khan H, Ali J. UHPLC/Q-TOF-MS: Introduction and Applications. Letters in Organic Chemistry. 2015; 12 (6): 371-378.

5.     S.C. Sweetmann, Ed. Martindal: The Complete Drug Reference, 33rd Ed. The Pharmaceutical Press, London, UK, 2002, pp. 322.

6.     S.C. Sweetmann, Ed. Martindal: The Complete Drug Reference, 33rd Ed. The Pharmaceutical Press London, UK, 2002, pp 332–333.

7.     K.D. Tripathi. Essentials of Medical Pharmacology, 4th Ed. Jaypee Brothers Medical, New Delhi, India, 1999, pp. 276–83.

8.     Wei Zhang, Futian Han, Harry Zhao, Zhongping Lin, Qingtao Huang and Naaidong Weng. Determination of metformin in rat plasma by HILIC-MS/MS combined with Tecan automation and direct injection. Biomedical chromatography, 2012, 26(10); 1163-1169.

9.     Wang Jian, Wang Ying-Wu, GU-Kai, Wu Ki, Wang Yan. Determination of metformin in human plasma by liquid chromatography-tandem mass spectrometric assay. Chem. Res. Chinese, 2005, 21(2), 246-250.

10.   Cristina georgita, Iulia sora, Florin Albu, Crina Maria Monciu. Comparison of a LC/MS method with a LC/UV method for determination of metformin in plasma samples. Farmacia, 2010, 58, 2,158-169.

11.   N. Yuzuak, T. Ozden, S. Eren, S. Ozilhan. Determination of glimepiride in human plasma by LC-MS-MS. Chromatographia, 2007, 66, S165-S168.

12.   Hohyun Kim, Kyu Young Chang, Hee Joo Lee, Sang Beom Han. Determination of glimepiride in human plasma by Liquid Chromatography-Electrospray Ionisation Tandem Mass Spectrometry. Bulletin of Korean Chemical Society, 2004, 25(1), 109-114.

13.   Deepti Jain1, Surendra Jain, Deepak Jain, and Maulik Amin. Simultaneous determination of Simultaneous Estimation of Metformin Hydrochloride, Pioglitazone Hydrochloride, and Glimepiride by RP-HPLC in Tablet Formulation. J. Chromatographic Science, 2008, 46, 501-504.

14.   N. Ramathilagam, P. Solairaj. Method development and validation of RP-HPLC method for glimepiride, metformin, and piogliazone in tablets. International Journal of Pharmacy and Analytical Research. 2014, 3(1), 46-54.

15.   Sengupta P., Bhoumik U., Ghosh A., Sarkar A.K. LC-MS-MS Development and validation for simultaneous quantization of metformin, glimepiride, and pioglitazone in human plasma and its application to bioequivalence study, Chromatographia, 2009, 69 (1), 1243-1250.

16.   Anandkumar R. Tengli, Shivakumar, B. M. UPLC-MS Method Development and Validation of Tablet Dosage form Containing Glimepiride, Metformin and Pioglitazone Using Internal Standard. IOSR Journal 0f Pharmacy, 2250-3013, 2319-4219.

17.   International Conference on Harmonization, ICH Q2 (R1), Validation of Analytical Procedures: Text and methodology, Geneva, 2005.

 

 

 

 

Received on 19.01.2017       Accepted on 12.02.2017     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2017; 7(1): 27-32.

DOI: 10.5958/2231-5713.2017.00005.8