Rapid Quantitative Estimation of Glipizide and Sitagliptin in Rat Plasma by Liquid Chromatography and Mass spectroscopy (LC-MS)

A. Anka Rao¹*, K. Pavan Kumar², A. Narayana Rao¹, Narender Malothu¹, B. Prassana Kumar Desu¹,

B. Naga Raju Bandaru¹

¹K L College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram,

Andhra Pradesh, India - 522 502.

²Research Scholar, K L College of Pharmacy, Koneru Lakshmaiah Education Foundation,

Vaddeswaram, Andhra Pradesh, India-522 502.

*Corresponding Author E-mail: drankarao83@gmail.com

ABSTRACT:

Objective: Highly responsive simple Liquid-liquid Extraction Glipizide development using deuterated Glipizide and Sitagliptin LC-MS/MS Glipizide for gradation of Glipizide and Sitagliptin in rat plasma Glipizide: The chromatographic condition involves, isocratic mode using waters X-Bridge C₁₈ 3.5µ (150x4.6mm) column. Mobile phase 0.1% orthophosphoric acid and Acetonitrile in the ratio (80:20) was used and detection was carried out in a positive mode of electrospray ionization by using MS. Results: Glipizide has been validated, the linearity was observed in the range 10-150 ng/ml and 0.1-1.5 ng/ml for Glipizide and Sitagliptin respectively. For intraday and inter-day precision %RSD values were found to be within the acceptable limits. Recovery studies for Glipizide and Sitagliptin obtained was 99.5% and 98.6% respectively. The battery of stability studies like bench-top, Autosampler stability, freeze-thaw, and long term stability was performed. Conclusion: Highly responsive simple liquid chromatography tandem mass spectroscopy assay Glipizidewas developed and witnessed for the gradation of Glipizideand Sitagliptin in rat plasma, the developed Glipizidewas applied to pharmacokinetic studies.

KEYWORDS: LCMS, Glipizide, Sitagliptin, Glipizidevalidation, pharmacokinetic study.

INTRODUCTION:

Oral anti diabetic drug used for treatment of type 2 diabetes and Glipizideis an antihyperglycemic agent and used for the management of type II diabetes. Currently, Glipizideis the first drug of choice for the management of type II diabetes. Glipizideconsidered as an antihyperglycemic drug because it lowers blood glucose concentrations in type II diabetes without causing hypoglycemia. Control high blood sugar levels helps to prevent kidney damage, blindness, nerve problems, loss of limbs, and sexual function problems.

Glipizide works by helping to restore your body's proper response to the insulin you naturally produce. Glipizide(Sitagliptin) is in a class of medications called sodium-glucose co-transporter 2 (SGLT2) inhibitors belongs to glifolins class and used for the treatment of type2 diabetes.^1,2,3

MATERIALS AND GLIPIZIDEHODS:

Chemicals and Reagents:

Acetonitrile (HPLC grade), Orthophosphoric acid (OPA) (Analytical grade) and water (HPLC grade) were purchased from Merck (India) Ltd., Worli, and Mumbai, India. All API’s of Glipizideand Sitagliptin as reference standards were procured from Spectrum Pharma research solutions Pvt. Ltd., Hyderabad. The combination of the formulation was procured from the local market.

Equipment:

HPLC system (Waters Alliance E2695 model) connected with mass spectroGlipizideer QTRAP 5500 triple quadruple instrument was used. Data processing was performed with Empower 2.0 software.

Chromatographic condition:

Chromatographic separation was carried out in an isocratic mode at room temperature using Waters X-Bridge C₁₈ (150x4.6mm, 3.5µ) column. The mixture of 0.1% OPA and Acetonitrile 80: 20v/v used as mobile phase and the flow rate was maintained at 1.0ml/min. The injection volume was 10µl and eluents were monitored at 258m/z using PDA detector. The run time was 5 min.

Preparation of Standard and Quality Control Samples:

Glipizide and Sitagliptin stock solutions were prepared for the calibration curve and quality control samples for validating the Glipizide hod and for subject sample analysis. Glipizide and Sitagliptin stock solutions were prepared in order to obtain the concentrations of 1000ng/ml and 10ng/ml respectively. From the stock solutions working standard and primary dilutions were prepared with diluent. Screening of blank rat plasma was carried out to before spiking to that it was free from any endogenous interference at the retention time of Glipizide and Sitagliptin. By spiking the blank plasma with appropriate amount of Glipizide and Sitagliptin seven point standard curve and four quality control samples were prepared. Samples calibration was made at concentrations of 10, 25, 50, 75, 100, 125, 150ng/ml of Glipizide 0.1, 0.25, 0.5, 0.75, 1, 1.25, and 1.5ng/ml of Sitagliptin .

Sample Preparation:

For sample preparation, 200µl of plasma sample, 300µl of Acetonitrile and 500µl of internal standard, 500µl of standard stock and 500µl of diluent to precipitate all the proteins and mix in the vortex cyclo mixture. Centrifuge at 500rpm for 30min. Collect the supernatant solution in the HPLC vial and inject it into the chromatogram.

Glipizidehod Validation:

Selectivity:

Analyzing six different rat plasma samples selectivity was performed for testing the interference of analytes at the retention times.

Matrix Effect:

Matrix effect for Glipizideand Sitagliptin was evaluated by comparing the peak area ratio in the post extracted plasma sample from 6 different drugs - free blank plasma samples and neat reconstitution samples. This experiment is performed in LQC, MQC and HQC levels in every three different preparations from the marketed formulation with six different lots of rat’s plasma. Finally, the recovery is within the acceptable limit (%CV) of ≤15%.

Precision and Acurracy:

It was done in six different quality control samples (n=6) from marketed formulation at a lower limit of quantification (LLOQ), low-quality control (LQC), medium quality control (MQC), high-quality control (HQC) levels are extracted to plasma. The %CV should be less than 15% for accuracy at LQC, MQC and HQC except LLOQ are should be within 20%.

Recovery:

The extraction efficiencies of Glipizideand Sitagliptin were determined by analysis of six replicates at each quality control concentration. The percentage recovery was evaluated by comparing the peak areas of extracted standards to the peak areas of unextracted standards.

Stability:

The stability of the samples is comparing the area response of analyte area response vs sample prepared from the freshly prepared sample solution. Stability studies were performed at the LQC and HQC concentration levels using six replicates at each level in plasma. In benchtop stability of spiked rat plasma samples stored at room temperature (benchtop stability) was evaluated for 24h. Autosampler Stability samples are spiked with rat plasma at LQC, MQC and HQC they are stored at 2-8°C in autosampler (autosampler stability) were evaluated for 24h. The autosampler stability was determined by comparing the extract plasma samples that were injected immediately, with the samples that were reinjected after storing in the autosampler at 2-8°C for 24h. The reinjection reproducibility was determined by comparing the extracted plasma samples that were injected immediately, with the samples that were reinjected after storing in the autosampler at 2-8°C for 24h. The freeze-thaw stability was conducted by comparing the stability samples that had been frozen at -30°C and thawed three times, with freshly spiked quality control samples. Six aliquots concentration each of LQC and HQC concentration levels were used for the freeze-thaw stability evaluation. Also, the studied drug showed stability in rat plasma when stored sample at -20°C for one month as long term stability when compared with the freshly prepared sample. As per US FDA guidelines, all stability conditions samples were stable below 15%.

RESULTS AND DISCUSSION:

In this Glipizidehas electrospray ionization (ESI) having maximum response over atmospheric pressure chemical ionization (APCI) mode has selected. Glipizide optimization of instrument to give sensitivity and signal stability during infusion of the analyte in the continuous flow of mobile phase to electrospray ion source operated at both polarities at a flow rate of 10µl/min. and ETR give more positive response in ion mode when compared to negative ion mode.

Trails have been performed to obtain the best chromatographic conditions with different columns such as C_18,C₈ and CN- propyl and mobile phases which are composed of 0.1% OPA and Acetonitrile were tested. Best chromatographic separation was occurred on X-Bridge C₁₈ column by using the mobile phase 0.1% OPA and Acetonitrile in 80:20 ratios at a flow rate of 1ml/min and detection was carried out at 258m/z by PDA.

Table 1: Within-run and between-run precision and accuracy for Glipizide

Nominal Conc. (ng/ml)	Within run			Between run
Nominal Conc. (ng/ml)	Mean (ng/ml)	Precision (%CV)	Accuracy	Mean (ng/ml)	Precision (%CV)	Accuracy
5	5.2452	0.53	99.7	5.1268	0.75	101.4
50	50.2369	0.67	97.6	50.2487	0.64	99.5
100	100.2635	0.72	100.2	100.2514	0.81	98.7
150	150.1578	1.69	98.2	150.1036	0.39	100.5

Table 2: Within-run and Between-run precision and accuracy for Glipizide

Nominal Conc. (ng/ml)	Within run			Between run
Nominal Conc. (ng/ml)	Mean (ng/ml)	Precision (%CV)	Accuracy	Mean (ng/ml)	Precision (%CV)	Accuracy
0.05	0.0568	0.95	100.5	0.05263	0.87	101.6
0.5	0.5247	0.65	100.7	0.5295	0.79	100.8
1	1.1658	0.52	99.8	1.1528	0.52	99.4
1.5	1.5263	0.46	99.5	1.5189	0.49	97.4

Linearity:

The peak area ratios of calibration standards in each assay over the nominal concentration range of 10-150ng/ml and 0.1-1.5ng/ml for Glipizide and Sitagliptin were observed respectively (Table No: 1). Linearity of calibration was described well by least square regression lines (fig No: 3 and 4). The correlation coefficient was ≥0.999 for Glipizide and Sitagliptin

Precision and Accuracy:

Polling all individual assay results of replicate of five separate batch runs have been analysed on four different days for inter-run precision and accuracy determination. The inter-run precision (%CV) was <5% and inter-run accuracy was between 95% and 105% for Glipizide and Sitagliptin. (Table .1 and 2)

Recovery:

Low, medium and high quality concentration levels of Glipizide and Sitagliptin fo Six aqueous (sample spiked reconstitution-solution) were prepared for recovery determination, and the areas obtained for extracted samples were analyzed with the same batch run on the same day. For Glipizide and Sitagliptin mean recovery was 99.79% and precision was 0.52%, which indicates the extraction efficiency for Glipizide and Sitagliptin.

Reinjection Reproducibility:

Reproducibilty of the samples was checked by performing back calculated concentration for reinjected samples and change was less than 2.0 at LQC and HQC concentrations. Sample was prepared to be reinjected after 24h, they also showed % change less than 2.0% at LQC and HQC concentration levels.

Stabilities:

Glipizide and Sitagliptin stock solution stability was performed by preparing in stock solutions with diluents and storing at 2-8°C in a refrigerator. Above stock solutions were compared with the stock solutions prepared 24 hours before. For Glipizide and Sitagliptin the percentage change observed was 1.27% and 0.75% respectively indicates the stock solutions were stable for at least 24 hours. Bench top and auto sampler stability for Glipizide and Sitagliptin was investigated at LQC and HQC levels.

Stability of Glipizide and Sitagliptin was not effected and was confirmed by repeated freezing and thawing (three cycle) of spiked plasma samples at LQC and HQC levels, they were stable in plasma for at least 24h at room temperature as well in an auto sampler at 20°C. In case of long term stability studies for Glipizide and Sitagliptin they were stable in the matrix for 24 hours at a temperature of -30°C. (Table 2 and 3).

Application To Pharmacokinetic Study:

LC-MS/MS Glipizide has developed and has been applied for the analysis of Glipizide and Sitagliptin in plasma samples obtained from drats and also applied to the pharmacokinetic studies. Glipizide and Sitagliptin were co-administered by oral gavage at a dose of 2.5 mg/kg and 0.0375 mg/kg respectively. The detailed pharmacokinetic para Glipizide (C _max, T_max, [AUC] _0–t and AUC_0–∞) of Glipizide and Sitagliptin are presented in 3.

Table 3: Mean pharmacokinetic paraGlipizideers of Glipizideand Sitagliptin

Pharmacokinetic parameters	Glipizide	Sitagliptin
AUC_0-t (ng h/ml)	337	106
C_max (ng/ml)	6	8.7
AUC_0-∞ (ng h/ml)	352	111
AUC_t-∞ (ng h/ml)	15	5
t_max (h)	23Hr	12

CONCLUSION:

Validated highly sensitive HPLC-ESI-MS/MS Glipizide has been developed for the determination of Glipizide and Sitagliptin simultaneous in rat plasma. Fast, rugged, reproducible simple bio analytical Glipizide has been developed which can be used in pharmacokinetic studies along with the monitoring of the investigated analyte in the body fluids. The proposed developed Glipizide is highly specific due to its inherent selectivity of tandem mass spectro Glipizide and has its significant prevalence over other described developed Glipizide. The proposed Glipizide can simultaneously estimate Glipizide and Sitagliptin in human plasma at a very low concentration in ng/mL. High recovery with liquid-liquid extraction Glipizide lesser retention time is time-saving when compared with other Glipizide. The specified Glipizide was simple, specific and rapid as well as allows for easy application in laboratories, valuable tool for bioavailability, bioequivalence and pharmacokinetic studies.

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Received on 05.06.2020 Modified on 18.01.2021

Research J. Pharm.and Tech 2022; 15(4):1675-1679.

DOI: 10.52711/0974-360X.2022.00280