Estimation of Gliclazide in Human Plasma by LCMS/MS

 

Srinivasa Reddy, Anandan Pattabi, Arvind Kumar, Sreedevi Rajpurohit, Arindam Mukhopadhyay* and Saral Thangam

Norwich Clinical Services Pvt. Ltd., 147/F, 8th Main, 3^rd Block, Koramangala, Bangalore– 560034

*Corresponding Author E-mail: arindam.mukhopadhyay@norwichclinical.com

Gliclazide and Glipizide (IS) were separated on a Reverse phase chromatography using the mobile phase mixture of ammonium acetate and methanol at a flow rate of 1.0 ml/min after extraction from plasma by liquid – liquid extraction method. Both the analyte and the IS were detected in API 4000 Mass spectrometer in the positive atmospheric pressure Ionization (API) mode with multiple reactions monitoring (MRM). The MRM transitions were monitored by following m/z for parent ion 324.1 and daughter ion 127.2 (Gliclazide), and m/z 446.1 and daughter 321.1 (Glipizide, IS). A linear calibration plot of Gliclazide was achieved in the concentration ranges of 7.776 ng/ml to 6000 ng/ml.  Mean recovery was 75.67%.This method was fully validated for specificity, precision, accuracy, reproducibility and other criteria as per regulations.

 

 

 

INTRODUCTION:

Gliclazide, an oral hypoglycemic drug, belongs to second-generation sulphonylureas. Chemically it is known as [1-(1-azabicyclo(3,3,0 octyl)-3-(p-tolylsulphonylurea)]. It lowers blood glucose concentration probably by reversing insulin resistance in type-II diabetic patients and improving defective insulin secretion. It also slows down the progression of diabetic angiopathy.  It is readily absorbed from the gastro-intestinal tract with peak concentrations   in plasma occurring about 2-4 h and it is highly protein bound [1–3].

 

Several analytical methods have been published for estimation of Gliclazide [4 – 9]. However these methods are either less sensitive, time consuming or non-validated to be used for drug estimation in samples from clinical trials. We are reporting here a simpler and sensitive method which is validated as per FDA regulations and can be used for analysis of Gliclazide during BA/BE studies.

 

 

Experimental:

Materials and Reagents:

Gliclazide (BP) was purchased from LGC Promochem, India, whereas Glipizide, used as an internal standard was from Vivan Life Sciences, India. HPLC grade methanol and n-hexane were obtained from E Merck (India). All other chemicals of highest purity grade were locally purchased. Milli Q water (Millipore (USA)) was used throughout the procedure.

 

K₂ EDTA containing human blood was collected in-house from healthy volunteers. Plasma was separated by centrifuging at 3000 rpm for 10 min at 4⁰C.

 

Preparation of Calibration standard samples:

Stock solutions (1mg/ml) of Gliclazide were prepared in methanol. Concentration was then corrected using the potency and actual amount weighed. Working solutions (388.800ng/ml to 300000.000ng/ml) were prepared by serial dilution of the stock solution by methanol: water (1:1, v/v).

 

Similarly for internal standard (IS), stock solution (1mg/ml) of Glipizide was prepared in methanol and corrected final concentration is obtained using the potency and amount weighed. Working solution (5µg/ml) was then prepared from this stock solution by serial dilution using methanol: water (1:1, v/v) as diluent. All solutions were stored in refrigerator at 2-8ºC.

 

Calibration standards of concentration range from7.776ng/ml to 6000.000ng/ml were prepared by adding 980µl of blank plasma to 20µl of respective working solution and stored at – 70ºC.

Preparation of Quality Control Samples:

Working solutions (395.600ng/ml to 230000.000ng/ml) were prepared by serial dilution of the stock analyte solution by methanol: water (1:1 v/v). Quality control samples, marked as LOQQC, LQC, MQC and HQC containing 7.912ng /ml, 23.000ng/ml, 2300.000ng/ml and 4600.000ng/ml of Gliclazide respectively, were prepared by adding 980µl of blank plasma to 20µl of respective working solution and stored at -70°C.

 

Sample extraction:

After thawing the sample at room temperature it was vortexed to ensure complete mixing of the contents. 100 µl of sample was added to each of the pre-labeled RIA vials except blank where only 100 µl of plasma is added. 50 µl of IS (Glipizide) was added to each of these analyte spiked plasma except blank. 100 µl of o.1 N HCl was added to each vial and vortexed. Then 2.5ml of extraction solvent mixture (diethyl ether: n-hexane:: 80:20, v/v) was added to each vial and kept on Vibramax at 2500 RPM for 10 min. It was then centrifuged at 4000 RPM for 10 min at 4°C. 2 ml of the supernatant was then transferred to new RIA vials and dried at 40°C in nitrogen evaporator. It was then reconstituted in 2 ml of mobile phase (10mM ammonium acetate pH 3.5±0.2: Methanol: 40:60 v/v) and vortexed. After centrifugation at 11000 RPM for 5 min the sample was transferred to a HPLC vial and loaded into the auto sampler. 10µl was injected to LCMS/MS for analysis.

 

Chromatography:

The drug was separated on a Zorbax SB C8 4.6 X 50 mm column with particle size 3.5µ (Agilent) using the mobile phase [10mM ammonium acetate (pH 3.5±0.2): Methanol:: 40:60 v/v] at a flow rate of 1.0 ml/min with a splitter in Waters UPLC attached to API 4000 Mass spectrometer (Applied Biosystems, USA) using  positive Atmospheric Pressure Ionization mode. The column oven temperature was maintained at 50⁰C and the run time was 2.5 min. The analyte and IS were detected on mass spectrometer operating in the multiple reaction monitoring (MRM) modes by following transitions at m/z 324.1 to127.2 (Gliclazide) and m/z 446.1 to 321.1 (Glipizide) with a dwell time of 200 msec. The API source temperature was maintained at 500⁰C. Data were acquired and processed with Analyst software 1.5.1.

 

Matrix Factor:

Six replicates of aqueous standard containing analyte at LQC concentration and the intended internal standard concentration and six replicates of aqueous standard containing analyte at HQC concentration and the intended internal standard concentration were injected to LCMS/MS.

Eight blank plasma (matrix) lots from different sources including one heamolysed and one lipemic were processed and then spiked with either LQC concentration of analyte and the intended internal standard concentration or HQC concentration of analyte and the intended internal standard concentration and processed as per the procedure mentioned earlier.

 

Individual analyte area response and IS area response of each post extracted sample were compared with the mean analyte area response and mean IS area response of the aqueous standard respectively. IS normalized method was used to calculate matrix factor. IS normalized matrix factor is obtained by substituting peak area with peak area ratio (analyte/IS) (Table 1).

 

Result and DISCUSSION:

Specificity and Selectivity:

For selectivity analysis, eight different lots of plasma including one hemolysed and one lipemic lots were spiked with analytes and internal standard. Interference at the retention times of analytes and IS was evaluated by comparing peak area response with that of blank plasma. Signal to noise ratio for all lots was more than 5.0 indicating the method is selective for Gliclazide. Retention times for Gliclazide and Glipizidewere 1.8min and 1.6min, respectively (Fig.1a and b). No interfering peaks were observed in the blank at the retention times corresponding to drug and IS indicating that the procedure is specific to Gliclazide.

Similarly, no matrix effect was found while analyzing the human plasma samples.

 

Fig. 1a. Gliclazide

 

Fig. 1b.  Glipizide

 

Table 1: Matrix Effect for estimation of Gliclazide

Matrix ID		Analyte Area In Absence of Matrix	Analyte Area in Presence of Matrix	Matrix Factor For Analyte	IS Area in Ab-sence of Matrix	IS Area in Presence of Matrix	Matrix Factor for IS
PL_243	24820		25808	105.26	2354838	2370855	98.85
PL_253	24330		25096	102.36	2305130	2368329	98.95
PL_256	24591		24647	100.53	2316292	2385823	98.23
PL_258	24793		24886	101.50	2345324	2343268	100.01
PL_259	24411		24384	99.45	2391100	2348618	99.78
PL_266	24162		23987	97.83	2348494	2339495	100.17
HPL_236			24113	98.35		2341431	100.09
LPL_212			24946	101.75		2376924	98.60

 

Linearity of the Calibration Plot:

Calibration plot (Fig 2) of Gliclazide showed that the calibrations are linear in the concentration ranges of 7.776  ng/ml to 6000 ng/ml with a correlation coefficient (r) of 0.99. Limit of detection was found to be 1ng/ml with a signal to noise ratio of more than 22.

 

Fig.2. Calibration plot

 

Precision and Accuracy:   

Intra- or inter- day precision and accuracy were determined by six replicate analysis of LOQQC, LQC, MQC and HQC samples. Intraday precisions were ranged from 2.45% to 5.73% for Gliclazide which were within acceptable limit (Table 2). Similarly, for inter-day batch precision ranged from 5.06% to 6.22% for Gliclazide was also within accepted limit (£ 20% at LOQQC and £15% for others).

 

Mean accuracy for intraday batch ranged from 99.68% to 106.78% for Gliclazide which are within acceptable limit (Table 2). Similarly, for inter-day batch accuracy ranged from 95.45% to 98.54% which were also within accepted limit (£ 20% at LOQQC and £15% for others).

 

Recovery:

Absolute recovery percentage was determined by comparing the mean peak area of Gliclazide obtained by injecting 6 extracted samples of LQC, MQC and HQC with the mean peak area obtained by injection of respective aqueous standard solutions. Mean percentage recovery was 75.67 with mean % CV of 5.29 (Table 3).

 

Stability:

Short – Term/bench - top stability

To check whether the sample is stable during analysis, six aliquots of LQC and HQC samples were thawed and kept at room temperature for 8.30 hours, which has been decided based on the time required for analysis. The samples were then processed and analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the freshly spiked samples indicating that Gliclazide was stable at room temperature (Table 4).

 

Auto sampler stability

The stability of the processed samples in the auto sampler during analysis was determined by using six aliquots of LQC, HQC samples.

 

 

Table 2: Accuracy and precision of analysis of Gliclazide in the quality-control samples

 

Table 3: Recovery of Gliclazide from biological matrix

 

The stability of Gliclazide was assessed for 16 hours, the expected run time for batches of validation samples. The result was then compared with that of freshly spiked samples. For IS in-injector stability, the IS/analyte area ratio of MQC stored in auto sampler for 16 hours was compared against freshly prepared MQC samples. No significant difference in the results indicated that the analyte and IS are stable for at least 16 hour in the auto sampler (Table 4).

 

Freeze –Thaw stability

Analyte stability was determined after four freeze thaw cycles for six aliquots of each of the LQC and HQC. The samples were stored below – 70⁰C for 24h and then allowed to thaw at room temperature unassisted. After complete thawing, the samples were again stored at same temperature (– 70⁰C) for 12h. The freeze thaw cycle was repeated another three times before analyzing the samples. No differences were noticed when the results were compared with the fresh QC samples indicating the stability of Gliclazide in K₂EDTA human plasma for four freeze thaw cycles (Table 4).

 

Wet Extract stability

To check whether the sample is stable after processing, six aliquots of LQC and HQC samples were processed and kept at room temperature for 2.5 hours. The samples were then analyzed as mentioned above. No significant differences were noticed when these results were compared with those obtained from the fresh QC samples indicating that processed samples of Gliclazide was stable at room temperature (Table 4).

 

Short term stock solution stability

To ensure that analyte is stable in appropriate solution for a short period of time at room temperature, the stability of stock and working solutions of Gliclazide was evaluated at room temperature for 25 hours. There were no significant changes in stabilities of the stock and working solutions on keeping at room temperature for 25 hours (Table 5).

 

Table 4: Stability of Gliclazide in Biological Matrix

Stability Check	Samples	Nominal Conc.	Observed Conc.	%cv	% Stability
Bench Top for (8.5hrs)	LQC (n=6)	23.015	24.9623	3.02	102.43
	HQC(n=6)	4794.720	5155.5002	4.24	103.46
Freeze Thaw (4 cycles)	LQC (n=6)	23.015	25.2852	3.90	103.75
	HQC(n=6)	4794.720	4986.4332	1.40	100.07
In-Auto-sampler(16hr)	LQC (n=6)	23.015	25.7227	1.85	103.31
	HQC(n=6)	4794.720	4965.6900	5.69	97.59
Wet Extract (2.5hr)	LQC (n=6)	23.015	23.9983	1.87	101.55
	HQC(n=6)	4794.720	4855.4952	1.98	99.67

 

 

Table 5: Stability of Aqueous GliclazideSolutions

Stability Check	Samples	Fresh Stock (Avg. Area)	Stability Stock (Avg. Area)	% Cv	% Stability
Short Term Stock Solution Stability (25hr)	Analyte(n=6)	1422245.3 (1106.0928µg/ml)	1423302.2 (1089.7092µg/ml)	1.89	101.58
	Internal Standard (n=6)	1940083.7 (1110.0746µg/ml)	1943931.7 (1083.7389µg/ml)	1.41	102.63
Short Term Working solution Stability (25hr)	Analyte (n=6)	1422245.3 (110609.280ng/ml)	1438430.5 (108970.920ng/ml)	1.76	102.66
	Internal Standard (n=6)	1940083.7 (5.2174µg/ml)	1966350.2 (5.0936µg/ml)	1.71	103.82
Long Term Stock Stability (16 Days)	Analyte(n=6)	1427089.2 (1106.0928µg/ml)	1285331.8 (1046.9520µg/ml)	1.01	95.15
	Internal Standard(n=6)	1921533.5 (1119.5157µg/ml)	2052374.2 (1147.3421µg/ml)	0.43	104.22

 

 

Long term stock solution stability

To evaluate the stability of analyte in appropriate solution for a long period of time under storage condition (2 – 8°C), the stability of stock solution of Gliclazide was evaluated at 2– 8°C for 16 days. Table 5 indicates that the stock solution was stable during the storage.

 

 

CONCLUSION

A simple, accurate, precise, sensitive and reproducible LCMS/MS method has been developed and validated for the determination of Gliclazide. The atmospheric pressure Ionization technique is used to overcome the matrix effect. The sample extraction procedure described here is LLE method which is not only a simpler method compared to other available extraction methods but also produces cleaner samples with no matrix effect. The method is applied successfully to quantify Gliclazide for determination of pharmacokinetic parameters in a BA/BE study.

 

ACKNOWLEDGEMENT:

The authors sincerely thank the management of Norwich Clinical Services for providing the opportunity to complete the project.

 

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Accepted on 04.07.2013         © RJPT All right reserved