Journal of Chromatography B, 965 (2014) 133–141

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Simultaneous determination of metformin and vildagliptin in human plasma by a HILIC–MS/MS method Roberto Pontarolo ∗ , Ana Carolina Gimenez, Thais Martins Guimarães de Francisco, Rômulo Pereira Ribeiro, Flávia Lada Degaut Pontes, João Cleverson Gasparetto Universidade Federal do Paraná, Department of Pharmacy, 632 Lothário Meissner Avenue, 80210-170, Curitiba – PR, Brazil

a r t i c l e

i n f o

Article history: Received 2 April 2014 Received in revised form 3 June 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Metformin Vildagliptin LC–MS/MS Human plasma HILIC

a b s t r a c t The objective of this work was to develop and validate a HILIC–MS/MS method for the simultaneous determination of metformin and vildagliptin in human plasma. Chromatographic separation was achieved using an Atlantis HILIC Silica 150-mm × 2.1-mm, 3-␮m particle size column maintained at 40 ◦ C. The isocratic mobile phase consisted of 20% water and 80% acetonitrile/water solution 95:5 (v/v), containing both 0.1% formic acid and 3 mM ammonium formate. The flow rate was maintained at 400 ␮L min−1 . Data from validation studies demonstrated that the new method is highly selective, sensitive (limits of detection 1 mL/min) [30,31]. In the latter cases, the use of high flow rates also increased the production of environmental waste or displaced the analytes into the dead volume. A faster method for monitoring metformin in plasma was achieved by laser diode thermal desorption/atmospheric pressure chemical ionization tandem mass spectrometry, a direct introduction technique where the analytes are desorbed into the mass spectrometer simultaneously without the use of chromatographic columns [29]. However, this system resulted in high variations of accuracy and precision, most likely due to ion suppression and/or matrix effects. Interest in HILIC–MS/MS methods has increased in the last few years because HILIC uses more organic solvent in the mobile phase than reversed-phase columns. It favors the ionization of the compounds and considerably increases the detection sensitivity [39]. In this way, the present method showed a limit of detection for metformin approximately 2 [16], 3 [9], 5 [14], 10 [12,23], 20 [19] and 40 [17] times lower than was obtained using HPLC–UV and at least 75 times lower than was obtained using capillary electrophoresis [6]. The new method also showed limits of detection and quantification for metformin similar to or lower than the limits obtained using other HPLC–MS methods available in the literature [28–34,42]. The HPLC–MS method available for vildagliptin [37] was developed only for qualitative purposes, while HPLC–UV [36] showed limits of detection and quantification 2000 times higher than the newly developed method. The new HILIC–MS/MS method was uniquely developed for the simultaneous quantification of vildagliptin and metformin in

137

human plasma and was also fully validated according to current worldwide regulations, thus ensuring reliable results. A fast run time was also achieved because mass spectrometry is a highly selective technique, and the hydrophilic column allowed excellent chromatography of vildagliptin and metformin (substances with distinct polarities) without using a gradient mobile phase. The linear range of detection was achieved with accuracy and precision, and low environmental waste was produced because the new HILIC–MS/MS method uses a low flow rate. The representative chromatograms for analytes and ISs are shown in Fig. 2.

3.2. Sample cleanup After method development, several extraction procedures were tested to achieve the best recovery of analytes and ISs, including solid-phase extraction (Oasis Hydrophilic-Lipophilic-Balanced (HLB) sorbent, Waters Corporation, Milford Ireland) and protein precipitation with acetonitrile and methanol (with or without formic acid 0.1%). Liquid–liquid extraction was not tested because organic solvents are incompatible with polar substances such as metformin. All the extraction procedures were performed by preparing QC samples in eight replicates at the MQC concentration level (Section 2.5). As a result, solid phase extraction (SPE) was not beneficial for extracting the analytes because the HLB sorbent retained vildagliptin (less polar substance), but did not efficiently retain metformin (polar substance). Thus, two sorbents would be necessary to obtain adequate recovery of the analytes, one polar (for extracting metformin) and the other nonpolar (for extracting vildagliptin), making sample cleanup expensive and laborious. Protein precipitation (PPT) was the only efficient technique for extracting metformin, vildagliptin and ISs. However, the proteins were not completely precipitated using methanol or methanol containing 0.1% formic acid. In these samples, variations in column pressure were observed, and consequently the samples could not be injected into the LC–MS/MS. The use of 100% acetonitrile and acetonitrile containing 0.1% formic acid as precipitating agent provided rapid precipitation of the proteins and formed a dense precipitate that was easily removed by centrifugation. In addition, acetonitrile containing 0.1% formic acid demonstrated the highest efficiency for extracting the analytes with adequate reproducibility (RSD < 5%) and was the first choice for sample cleanup.

3.3. Method validation 3.3.1. Limits of detection (LOD) and lower limits of quantification (LLOQ) The high sensitivity of the developed method was demonstrated by the low LOD (signal-to-noise ≥3), estimated to be 1.5 ng mL−1 for metformin and 0.75 ng mL−1 for vildagliptin. The LLOQ, based on a signal-to-noise ratio ≥10 with appropriate precision (RSD < 10%), was estimated to be 5.0 ng mL−1 for both metformin and vildagliptin.

3.3.2. Selectivity The chromatograms obtained using normal, hemolyzed, lipemic and spiked plasmas are presented in Fig. 3. This method did not reveal additional endogenous interference peaks at the retention times of the ISs. Potential interfering peaks in the biological matrix appeared near the retention times of metformin and vildagliptin. However, the interfering peaks are at noise levels and did not exceed 5% of the response of the analyte in the LLOQ concentration. The developed method was therefore considered to be selective.

138

R. Pontarolo et al. / J. Chromatogr. B 965 (2014) 133–141

Fig. 2. Representative LC–MS/MS chromatograms of human plasma spiked with standards of metformin (Tr: 2.94 min), vildagliptin (Tr: 2.56 min) and the internal standards metformin-related compound B (2.90 min) and pyrantel (2.21 min). Data: positive ion mode; sample cleanup performed through protein precipitation using acetonitrile 0.1% formic acid; signals not smoothed.

3.3.3. Linearity After three days of assessment, the calibration curves of metformin and vildagliptin showed excellent linearity with correlation coefficients (r) > 0.99. The means of the individual linear equations and correlation coefficients were as follows: metformin, y = 11.2x + −0.0319 (r = 0.9966) and vildagliptin, y = 7.49x + 0.0222 (r = 0.9973). At all concentration levels, the variations in precision (RSD%) were less than 10%, with values ranging from 0.5 to 7.1% for metformin and from 0.6 to 8.1% for vildagliptin. The individual values for accuracy at each concentration level ranged from 88.10 to 110.0% for metformin and from 86.3 to 110.0% for vildagliptin. These results guaranteed a reliable response, regardless of the concentrations utilized.

3.3.4. Precision, accuracy and sample dilution The results for the accuracy and precision of the analyses are shown in Table 2. The new method was precise for all the analytes and ISs, with RSDs varying from 1.85 to 7.05% for intra-day and from 3.5 to 9.01% for inter-day analysis. The method also showed satisfactory accuracy, with relative errors ranging from −1.88 to 9.50% for intra-day and from −4.32 to 11.15% for inter-day analysis. The analyses also demonstrated that samples subjected to dilution exhibited the same precision and accuracy as undiluted samples (Table 2). Therefore, the developed method was considered to be reproducible and accurate.

3.3.5. Carryover test With alternate injections of the highest QC evaluated level and blank plasma samples, no significant interfering peaks were observed at the retention times of the analytes and ISs. There was therefore no risk of carryover contamination between injections. 3.3.6. Extraction recovery and matrix effect The results of the matrix effect and the individual extraction recoveries of the analytes and ISs are presented in Table 3. The variations of the normalized effects of matrix of each compound were less than 15%, indicating that the effects of the biological matrix on the response of the analytes and ISs were insignificant. For all compounds, satisfactory recovery (>75%) was achieved with high reproducibility (RSD < 4.0%). Protein precipitation with acetonitrile containing 0.1% formic acid was therefore considered effective for extracting metformin and vildagliptin from human plasma. 3.3.7. Ion suppression/enhancement The extent of ion suppression or enhancement was investigated to evaluate not only inferences by the matrix but also interferences by co-eluting analytes and ISs. As demonstrated in Table 4, the p values >0.05 indicated that the mean peak areas of analytes or ISs injected alone are similar to the values obtained with co-eluted samples. The extents of ion suppression

R. Pontarolo et al. / J. Chromatogr. B 965 (2014) 133–141

139

Fig. 3. Chromatograms obtained by HILIC–MS/MS for the selectivity study. Positive ion mode: (A) normal blank plasma; (B) lipemic blank plasma; (C) hemolyzed blank plasma; (D) normal blank plasma spiked with metformin (5.0 ng mL−1 ), vildagliptin (5.0 ng mL−1 ) and ISs metformin-related compound B (350.0 ng mL−1 ) and pyrantel (250.0 ng mL−1 ).

and ion enhancement effects were lower than 7.89%. Therefore, no significant ion enhancement or suppression occurred by coeluting analytes and ISs, and these effects do not influence drug quantification.

3.3.8. Stability assay Table 5 summarizes the mean recovery of the analytes and ISs obtained after a variety of storage and handling conditions. Under the conditions evaluated, an RSD and RE < 15% demonstrated no

Table 2 Precision and accuracy of metformin (MET), vildagliptin (VILD), metforminrelated compound B (METB) and pyrantel (PYR) obtained in human plasma by HILICMS/MS experiments. Compounds

Quality Control level

Precision

Accuracy Standard concentration (ng mL−1 )

Intra-day RE%

Inter-day RE%

Intra-day (RSD%)

Inter-day (RSD%)

MET

LLOQ LQC MQC HQC DQC

5.0 12.5 100.0 375.0 1000.0

9.50 −4.24 −2.12 7.88 −1.88

11.15 0.03 3.24 4.74 2.56

5.93 7.05 4.74 2.37 2.72

8.80 9.01 7.99 8.85 6.56

VILD

LLOQ LQC MQC HQC DQC

5.0 12.5 100.0 375.0 1000.0

−3.35 6.77 3.67 5.60 −2.32

2.53 10.06 8.68 −5.23 5.57

1.85 3.42 2.72 3.43 3.31

3.50 3.73 6.06 5.18 6.32

METB* PYR*

– –

350.0 250.0

5.31 −7.45

3.39 −4.32

2.89 2.22

7.45 3.63

LLOQ: lower limit of quantification; LQC: low quality control; MQC: medium quality control; HQC: high quality control; DQC: dilution quality control; intra-day analysis, n = 8; inter-day analysis, n = 24; RE%: relative error; RSD%: relative standard deviation; *internal standard.

140

R. Pontarolo et al. / J. Chromatogr. B 965 (2014) 133–141

Table 3 Extraction recovery for metformin (MET), vildagliptin (VILD), metformin-related compound B (METB) and pyrantel (PYR) in human plasma samples using protein precipitation with acetonitrile containing 0.1% formic acid. Matrix effect

Recovery

Compounds

Added concentration (ng mL−1 )

Recovery concentration (ng mL−1 ± SD%)

MET

12.5 375.0

11.63 ± 0.22 334.50 ± 8.80

VILD

12.5 375.0

13.41 ± 0.20 387.08 ± 11.53

METB* PYR*

350.0 250.0

340.73 ± 6.50 189.90 ± 7.29

Extraction precision (RSD%)

NEM

Mean ± SD

NEM (RSD%)

93.0 89.2

1.89 2.65

0.0891 0.0877

0.0884 ± 0.0009

1.04

107.3 103.2

1.49 2.98

1.1799 1.2414

1.2107 ± 0.0435

3.59

97.3 76.0

1.91 3.83

N/A N/A

N/A N/A

N/A N/A

Mean recovery (%)

SD: standard deviation; RSD%: relative standard deviation; *internal standard; NA, not applicable.

Table 4 Co-eluting analyte pairs and the relative extents of ion suppression or enhancement effects measured with the new HILIC–MS/MS method. Co-eluting analyte pair

Relative extent of ion suppression or enhancement effects of

Analyte (A)

Internal Standard (B)

A influenced by B (mean % ± RSD, p value*)

B influenced by A (mean % ± RSD, p value*)

Metformin

Metformin related compound B Pyrantel

−2.54 ± 4.53 (p = 0.2880)

−7.89 ± 3.84 (p = 0.1081)

3.23 ± 2.48 (p = 0.2011)

−4.96 ± 2.22 (p = 0.1556)

Vildagliptin

Data: IS: internal standard; RSD%: relative standard deviation; *two-tailed Student’s t-test (95% confidence)

Table 5 Stability data for metformin, vildagliptin, metformin compound B and pyrantel under various storage conditions (n = 8). Stability

6 h at room temperature

15 days at 4 ◦ C

Short-term

Freeze–thaw cycles

Long-term

Postpreparative

Metformin

Vildagliptin

Metformin compound B*

Pyrantel*

Level 12.5 (ng mL−1 )

Level 375.0 (ng mL−1 )

Level 12.5 (ng mL−1 )

Level 375.0 (ng mL−1 )

Level 350.0 (ng mL−1 )

Level 250.0 (ng mL−1

Mean recovery (ng mL−1 ± SD)

12.4 ± 0.1

367.2 ± 5.4

12.4 ± 0.1

348.9 ± 17.7

345.9 ± 2.9

244.5 ± 3.8

RSD (%) RE (%)

0.78 −0.48

1.48 −2.07

0.73 −1.03

1.78 −2.48

0.84 −1.18

1.57 −2.19

Mean recovery (ng mL−1 ± SD)

11.8 ± 0.5

345.2 ± 20.2

12.1 ± 0.2

348.9 ± 17.7

338.69 ± 12.3

230.5 ± 13.2

RSD (%) RE (%)

3.86 −5.31

5.85 −7.95

2.09 −2.91

5.09 −6.95

2.32 −3.23

5.74 −7.80

Mean recovery (ng mL−1 ± SD) RSD (%) RE (%)

13.2 ± 0.2

341.5 ± 21.2

12.2 ± 0.3

355.1 ± 17.1

337.1 ± 11.4

260.7 ± 9.6

1.52 5.60

6.21 −8.93

2.62 −2.4

4.82 −5.3

3.38 −3.69

3.68 4.28

Mean recovery (ng mL−1 ± SD)

12.8 ± 0.2

381.3 ± 4.5

12.7 ± 0.2

383.18 ± 5.8

NA

NA

RSD (%) RE (%)

1.84 2.64

1.19 1.69

1.34 1.91

1.52 2.18

NA NA

NA NA

Mean recovery (ng mL−1 ± SD) RSD (%) RE (%)

11.9 ± 0.4

354.82 ± 16.8

12.1 ± 0.3

377.4 ± 10.3

NA

NA

3.36 −4.8

4.73 −5.38

3.63 −3.2

2.73 0.64

NA NA

NA NA

Mean recovery (ng mL−1 ± SD)

11.0 ± 0.4

331.1 ± 6.7

11.3 ± 0.1

325.6 ± 0.3

307.2 ± 6.1

218.2 ± 5.1

RSD (%) RE (%)

3.61 −10.2

2.02 −11.71

0.88 −9.94

0.95 −6.98

1.97 −12.23

2.35 −12.72

SD: standard deviation; RSD%: relative standard deviation; RE%: relative error; NA: not applicable; *internal standard.

R. Pontarolo et al. / J. Chromatogr. B 965 (2014) 133–141

141

Table 6 Amounts of metformin and vildagliptin in human plasma volunteers after administration of different drugs. Time points (h)

0 1 2 3

Medicines Glifage®

Galvus®

Galvus Met®

Metformin ng mL−1 ± SD

Vildagliptin ng mL−1 ± SD

Metformin ng mL−1 ± SD

Vildagliptin ng mL−1 ± SD

0.00 110.0 ± 12.21 122.3 ± 11.02 108.3 ± 7.51

0.00 6.78 ± 1.48 23.2 ± 5.38 32.8 ± 8.96

0.00 114.0 ± 9.90 220.0 ± 7.07 265.5 ± 41.71

0.00 11.1 ± 2.69 22.9 ± 4.95 22.7 ± 5.44

n = 6; SD: standard deviation; Available tablet formulations: Glifage® (metformin hydrochloride 850 mg), Galvus® (vildagliptin 50 mg) and Galvus Met® (vildagliptin 50 mg + metformin hydrochloride 850 mg).

significant differences between the amounts obtained with freshly prepared samples and with stored samples. Thus, excellent stability was shown for all compounds. 3.4. Method application The newly developed and validated HILIC–MS/MS method was applied to the determination of metformin and vildagliptin in human plasma. The results are shown in Table 6. As demonstrated, at three time points of blood collection, the new method successfully determined metformin and vildagliptin in samples of human volunteers who orally received a single dose of Glifage® (metformin hydrochloride 850 mg), Galvus® (vildagliptin 50 mg) and Galvus Met® (vildagliptin 50 mg + metformin hydrochloride 850 mg). The results demonstrated the presence of distinct levels of the substances from the plasma of patients, showing individual variability. The new method proved to be able to quantify the analytes under real samples and can be used as a tool for clinical monitoring of metformin and vildagliptin. 4. Conclusion A new, reproducible, sensitive, fast and fully validated HILIC–MS/MS method was developed for the determination of metformin and vildagliptin in human plasma. Data from the validation study demonstrated that the method is selective, linear, precise, accurate and free of residual and matrix effects. Under normal working conditions, excellent stability was shown for all compounds. The newly developed method was successfully applied to real samples and was found to be suitable for the quantification of metformin and vildagliptin in human plasma following the oral administration of tablets containing these substances. The new method proved to be suitable for clinical monitoring of metformin and vildagliptin. Conflict of interest The authors have declared no conflicts of interest. Acknowledgements The authors would like to thank the Ministério da Ciência e Tecnologia – MCT, Ministério da Saúde, Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (MCT/CNPq/CTSAÚDE/MS/SCTIE/DECIT) and Secretaria da Ciência, Tecnologia e Ensino Superior (SETI-PR) for the financial support of laboratory infrastructure. References [1] A.D. Assoc, Diab. Care 34 (2011) S62–S69. [2] D. Nathan, J.B. Buse, M.B. Davidson, E. Ferrannini, R.R. Holman, R. Sherwin, B. Zinman, Diabetologia 52 (2009) 17–30.

[3] I.W. Campbell, H.C.S. Howlett, Diab. Metab. Res. Rev. 11 (1995) S57–S62. [4] E. Ferrannini, V. Fonseca, B. Zinman, D. Matthews, B. Ahren, S. Byiers, Q. Shao, S. Dejager, Diab. Obes. Metab. 11 (2009) 157–166. [5] D.R. Matthews, S. Dejager, B. Ahren, V. Fonseca, E. Ferrannini, A. Couturier, J.E. Foley, B. Zinman, Diab. Obes. Metab. 12 (2010) 780–789. [6] J.Z. Song, H.F. Chen, S.J. Tian, Z.P. Sun, J. Chromatogr. B 708 (1998) 277–283. [7] J. Brohon, M. Noel, J. Chromatogr. 146 (1978) 148–151. [8] M.S. Lennard, C. Casey, G.T. Tucker, H.F. Woods, Br. J. Clin. Pharm. 6 (1978) 183–185. [9] H. Amini, A. Ahmadiani, P. Gazerani, J. Chromatogr. B 824 (2005) 319–322. [10] C.L. Cheng, C.H. Chou, J. Chromatogr. B 762 (2001) 51–58. [11] C. Yardimci, N. Ozaltin, A. Gurlek, Talanta 72 (2007) 1416–1422. [12] K.H. Yuen, K.K. Peh, J. Chromatogr. B 710 (1998) 243–246. [13] K.M. Huttunen, J. Rautio, J. Leppanen, J. Vepsalainen, P. Keski-Rahkonen, J. Pharm. Biomed. Anal. 50 (2009) 469–474. [14] F. Tache, V. David, A. Farca, A. Medvedovici, Microchem. J. 68 (2001) 13–19. [15] F.A. Siddiquia, N. Sherb, N. Shafia, S.S. Bahadurb, Arabian J. Chem. (2013), in press. [16] Q.F. Liu, Z.D. Li, X.J. Shi, Z. Jiao, M.K. Zhong, Chromatographia 70 (2009) 1511–1514. [17] H.P. Chhetri, P. Thapaa, A.V. Schepdaelb, Saudi Pharm. J. (2013), in press. [18] G. Gopi, M. Manikandan, D.N. Roja, S. Thirumurugu, K. Kannan, D.C. Arumainayagam, R. Manavalan, J. Pharm. Sci. Res. 4 (2012) 1676. [19] A. Rashid, M. Ahmad, M.U. Minhas, I.J. Hassan, M.Z. Malik, Pakistan J. Pharm. Sci. 27 (2014) 153–159. [20] D. Bhavesh, G. Chetan, K.M. Bhat, Shivprakash, Indian J. Pharm. Edu. Res. 41 (2007) 135–139. [21] M.C. Ranetti, M. Ionescu, L. Hinescu, E. Ionica, V. Anuta, A.E. Ranetti, C.E. Stecoza, C. Mircioiu, Farmacia 57 (2009) 728–735. [22] R.Q. Gabr, R.S. Padwal, D.R. Brocks, J. Pharm. Pharm. Sci. 13 (2010) 486–494. [23] A. Zarghi, S.M. Foroutan, A. Shafaati, A. Khoddam, J. Pharm. Biomed. Anal. 31 (2003) 197–200. [24] V. Porta, S.G. Schramm, E.K. Kano, E.E. Koono, Y.P. Armando, K. Fukuda, C.H.R. Serra, J. Pharm. Biomed. Anal. 46 (2008) 143–147. [25] X.Y. Chen, Q. Gu, F. Qiu, D.F. Zhong, J. Chromatogr. B 802 (2004) 377–381. [26] M.A.S. Marques, A.D. Soares, O.W. Pinto, P.T.W. Barroso, D.P. Pinto, M. Ferreira, E. Werneck-Barroso, J. Chromatogr. B 852 (2007) 308–316. [27] N. Koseki, H. Kawashita, M. Niina, Y. Nagae, N. Masuda, J. Pharm. Biomed. Anal. 36 (2005) 1063–1072. [28] A. Liu, S.P. Coleman, J. Chromatogr. B 877 (2009) 3695–3700. [29] J.G. Swales, R. Gallagher, R.M. Peter, J. Pharm. Biomed. Anal. 53 (2010) 740–744. [30] S.L. Bonde, R.P. Bhadane, A. Gaikwad, D. Katale, S. Gavali, A.S. Narendiran, Int. J. Pharm. Pharm. Sci. 5 (2013) 463–470. [31] S.R. Polagania, N.R. Pillib, R. Gajulab, V. Gandu, J. Pharm. Anal. 3 (2013) 9–19. [32] C. Georgita, F. Albu, V. David, A. Medvedovici, J. Chromatogr. B 854 (2007) 211–218. [33] C. Georgitã, I. Sora, F. Albu, C.M. Monciu, Farmacia 58 (2010) 158–169. [34] L.Y. Chen, Z.F. Zhou, M. Shen, A.D. Ma, J. Chromatogr. Sci. 49 (2011) 94–100. [35] N. Li, Y. Deng, F. Qin, J. Yu, F.M. Li, Biomed. Chromatogr. 27 (2013) 191–196. [36] A.B. Pharne, B. Santhakumari, A.S. Ghemud, H.K. Jain, M.J. Kulkarni, Int. J. Pharm. Pharm. Sci. 4 (2012) 119–123. [37] C. Hess, F. Musshoff, B. Madea, Anal. Bioanal. Chem. 400 (2011) 33–41. [38] FDA, Bioanalytical Method Validation, Guidance for Industry Bioanalytical Method Validation, U. S. Department of Health and Human Service, Food and Drug Administration, 2013. [39] Y.Z. Yang, R.I. Boysen, M.T.W. Hearn, J. Chromatogr. A 1216 (2009) 5518–5524. [40] A.J. Alpert, J. Chromatogr. 499 (1990) 177–196. [41] D. Remane, M.R. Meyer, D.K. Wissenbach, H.H. Maurer, Rapid Comm. Mass Spec. 24 (2010) 3103–3108. [42] L. Zhang, Y. Tian, Z.J. Zhang, Y. Chen, J. Chromatogr. B 854 (2007) 91–98.

MS method.

The objective of this work was to develop and validate a HILIC-MS/MS method for the simultaneous determination of metformin and vildagliptin in human ...
967KB Sizes 0 Downloads 5 Views