INTRODUCTION:

Esomeprazole (fig.1) is the first proton pump inhibitor (PPI) developed as an optical isomer (S-Omeprazole) for the treatment of acid-related disease⁽¹⁾.Esomeprazole is a potent inhibitor of gastric acid secretion and accumulates in the acidic compartment of the parietal cells where the molecule is transformed to its active sulfonamide form. Esomeprazole does not undergo chiral inversion in vivo ⁽²⁾and therefore esomeprazole can be determined using the same methodology as for its racemate, omeprazole. Omeprazole has been determined in blood plasma by liquid chromatography with UV-detection^(3,4) and this technique has also been employed for simultaneous assay of the two major metabolites. In recent years the combination of liquid chromatography and mass spectroscopy (LC-MS) has been used for omeprazole and metabolites^(5,6) and for omeprazole and other PPIs ⁽⁷⁾.

The aim of work reported in this paper was developed and validated a simple, highly sensitive and selective LC-MS/MS method for quantitative analysis of esomeprazole in human plasma.

In this paper we present a method based on LC-MS/MS after liquid – liquid extraction of esomeprazole with rabeprazole (Fig-2) used as an internal standard. Plasma volume is 80 µl and a liquid chromatography run time is about 2.2 min.

Chemicals and Reagents:

Working standard of Esomeprazole (Purity 99.61%) was obtained from Smilax laboratories Ltd., (Hyderabad, India). The internal standard Rabeprazole (Purity 99.52%) was obtained from Nifty Labs (Hyderabad, India). Acetonitrile was obtained from Merck (Mumbai, India). Methanol was obtained from LobaChemie (Mumbai, India). Ethyl acetate, Diethyl amines were obtained from Qualigens (Mumbai, India). All solvents and chemicals were HPLC or analytical grade. Ultra-pure analytical grade type – I water for HPLC was produced by milliQ plus water system (Millipore Corporation, Bedford, MF, USA) and used for the preparation of the sample and aqueous solutions. Plasma was obtained from Azidus Laboratories, Chennai.

Instrumentation:

Chromatographic analysis was carried out on a Waters alliance 2695 separation module with 2487 – Dual λ Absorbance detector and mass spectrometer on a Micromass quattro micro^TM API with data anlaysis software masslynx version 4.1(Milliford, MA). Samples were stored in a – 86⁰C ultra low deep freezer (Sanyo (Leicestershire,UK) at -70⁰C,

Fig.1.Structure of Esomeprazole

Fig.2.Structure of Rabeprazole

Stock and Working Solution Preparation:

Standard stock solution of Esomeprazole and Rabeprazole solution were prepared in methanol. Esomeprazole and Rabeprazole were weighed on a mettler toledo MX5 analytical balance (Mettler Toledo, Inc, Hightstown, NJ, USA). Appropriate amounts of drugs were dissolved using methanol in volumetric flasks to make a 1 mg / ml stock solution each. The stock solution of Esomeprazole was further diluted with a methanol containing 0.02% Diethylamine in order to obtain eight working solution for calibration standards as 100, 200, 500, 1600, 4000, 10000, 17000 and 20000 ng/ml. The quality control (QC) samples were prepared by diluting stock solution in methanol containing 0.02% Diethylamine to obtain the final concentration of 100, 300, 2200, and 16000 ng/ml. The Internal standard was prepared from 1 mg/ml stock solution by diluting with methanol containing 0.02% Diethylamine. All the solution were stored at 2-8⁰C.

Calibration Standards and QC Sample Preparation:

Calibration curve samples were freshly prepared by adding 50 µl of working standard solution to 950 µl blank plasma to yield a concentration of 5, 10, 25, 80, 200, 500, 850 and 1000 ng/ml of Esomeprazole. The QC samples were prepared adding 50 µl preparative QC solution to 950 µl of blank plasma to yield a concentration of 5, 15, 110 and 800 ng/ml of Esomeprazole. All stock solution were stored in -86⁰C ultra low deep freezer at -70⁰C until analysis.

Sample Preparation:

Extraction of Esomeprazole and Rabeprazole was carried out by liquid – liquid extraction. The extraction procedure was validated by spiking human plasma with known concentration of Esomeprazole and internal standard. Human plasma (80 µl) was transferred into poly propylene RIA vial and 50 µl of I.S. was added 200 µl of sodium bicarbonate (100 mm) was added for alkalinity. The mixture briefly vortexted by Vibromax Shaker mixer and 2 ml of Ethylacetate was added. The mixture was extracted by Vibromax Shaker for 15 mins and centrifuged at 4000 rpm for 15 mins. The upper organic layer was pipetted into clear tube and placed into the low volume evaporator at 40⁰C under the stream of N₂ gas(15 PSI) until dry. The dried residue was reconstituted in 200 µl of water: 0.02% DEA in methanol (3:7 v/v/) mixture an aliquot of 10 µl was injected into the LCMS / MS system.

Chromatography and Mass spectrometry Condition:

The mobile phase used for the analysis consist of Acetonitrile : water (pH ,7.0 with Ammonia) [8:2v/v]. The mobile phase was filtered through 0.2µ membrane filter before being used to prevent entry of bubbles or impurities in the system. The solution was degassed and sonicated with vaccum for approximately 15 mins before use. The mobile phase was delivered at a flow rate of 1.0 ml/min with 1:1 split. The analysis was carried out using Gemini C18, 50mmX4.6mm, 5m (phenomex, USA) HPLC column maintained at 30⁰C throughout experiment.

Electrospray ionization (ESI) with multiple reaction monitoring (MRM) was used to acquire mass spectra. Ions were monitored in positive mode and ion signals measured for Esomeprazole were m/z 346-31à 198.22 and internal standard were m/z 360.21 à 242.3 respectively. Quantitation of the analytes in human plasma was based on the detector response ratio of Esomeparazole versus internal standard.

Method validation :

The method validation assays were carried out by following the currently accepted US food and drugs administration (FDA) Bioanalytical method validation guidance. ⁽⁸⁾

Specificity and Selectivity:

Six human blood samples with six individual donors receiving no medication were extracted and analyzed for the assessment of potential interferences with endogenous substances. Two replicates from each of those six human plasma of Esomeprazole / Rabeprazole free blank human plasma samples were spiked with Esomeprazole and Rabeprazole. And then extracted along with two replicates from each of those six human plasma of Esomeprazole / Rabeprazole free blank human plasma samples without spiking Esomeprazole /Rabeprazole.

Matrix Effect:

The matrix effect on the ionization of analytes was evaluated by comparing the peak area of analyte resolved in blank samples (the final solution of blank plasma after extraction and reconstitution) with that resolved in mobile phase. Three different concentration level, LQC, MQC, and HQC (15, 110 and 800 ng/ml) of Esomeprazole and 50 ng/ml of I.S were evaluated by analyzing six samples at each level. The blank plasma used in this study was six different batches of healthy human blank plasma. If the ratio ±15% an exogenous matrix effect was impute.

Precision and Accuracy:

The validated method has been found to be reproducible by performing three precision and accuracy consisting of the one intra day batch and two inter day batches. Each analytical run in precision and accuracy consist of two replicates standards at LLOQ and ULOQ, and one replicates at other levels along with six replicates of QC at all levels.

Intraday run is evaluated from the precision and accuracy of six replicates of QC samples at LLOQ, LQC, MQC and HQC levels from the first three accepted analytical runs individually.

Interday run is evaluated from the precision and accuracy of 6 replicates of QC samples at LLQC, LQC, MQC, and HQC levels obtained from the first three accepted analytical runs.

Recovery:

The extraction efficiency of Esomeprazole was evaluated in human plasma by comparing six replicates of extracted QC samples at LQC, MQC and HQC level against unextracted samples containing equivalent of 15, 110, 800 ng/ml at the end of extraction. The recovery of I.S was evaluated by comparing the mean detector response of six plasma samples of mean detector response of standard solution of the I.S at similar concentration. As per the acceptance criteria the recovery of the analyte need not be 100% but the extent of recovery of an analyte should be consistent, precise and reproducible.

Stability:

Freeze thaw stability:

Effect of freeze and thaw cycles on stability of plasma samples containing Esomeprazole was determined by subjecting six aliquots of low (15 ng/m1) and high (800 ng/ml). Unprocessed QC sample were stored at -20⁰C to three freeze thaw cycles. After the completion of third cycle, the sample were analyzed. The samples were to be concluded stable in the % change in concentration of the stability samples within ±15% of the actual value.

Bench top stability:

Stability of Esomeprazole was evaluated in Bench top conditions over a period of 6 hrs at ambient temperature. Samples were prepared at LQC, HQC levels and kept on working bench for 6 hrs before analysis. Analysis was done in pent plate at each concentration levels.

Auto injector stability:

Stability of samples in auto injector was carried out for over a period of 68 hrs and it was evaluated by injecting QC samples (15,110, and 800 ng/ml).The stability was carried over a period of 68 hrs.

Long term stock solution stability:

For determining the stock solution stability of Esomeprazole working solution of 110 ng/ml were kept at -20⁰C for 23 days and it was is compared against the response obtained from six replicates of aqueous solutions of MQC (110 ng/ml) levels prepared from freshly prepared stock solution of Esomeprazole.

Long term frozen stability:

Six aliquot each of low (15 ng/mg) and high (800 ng/mg) QC samples were kept in deep freezer at -70⁰C for 29 days. There after the samples were preceded and analyzed along with precision and accuracy batch the concentrations thus obtained were compared with nominal values. All the values within ±15.0% of the nominal concentration qualified the test.

RESULT AND DISCUSSION:

The LC-MS/MS method for the detection of Esomeprazole in human plasma was investigated (Fig.3) shows the full scan Q₁, mass spectrum of Esomeprazole, where the molecular ion (MH⁺) was m/z 346.31. The daughter ion spectrum of Esomeprazole (Fig4) illustrator two major peaks m/z 198.22 and 151. 31. The full scan Q₁, mass spectrum and the daughter ion spectrum of I.S (Fig.5and6) displayed a patten. The protonated molecular ion (MH⁺) was m/z 360.21 and the base peak at daughter ion spectrum was m/z 242.30. MRM ion chromatography were used to determine Esomeprazole and I.S levels in Human plasma. The ions monitored for Esomeprazole were m/z 346.31 and 198.22, those for the I.S were m/z 360.21 and 242.30. The dwell time was 100ms for each ion with a 10ms pulse time between scans. Esomeprazole and I.S elevated at 0.66 min respectively. Relatively short run time, 2.2 min, was achieved with the short analytical column Gemini (50 X 4.6 mm I.D, 5µ).

Fig.3.Esomeprazole Parent ion Spectrum

At the initial stage of method development, both an ionspray interface and a heated nebulizer probe with corona discharge chemical ionization (APCI Interface) were evaluated. The APCI interface was choosen owing to its consistent response to the analytes in the MRM mode by adjusting the resolutions (LM1 and HM1) and peak width (delta mass). The instrument selectivity and sensitivity were greatly enhanced.

Fig.4.Esomeprazole Daughter ion Scan

Fig.5.Rabeprazole Parent ion Scan

Fig.6.Rabeprazole Daughter ion Scan

Chromatogram of Human blank control plasma fortified with I.S (Fig.7) Six lots of commercial blank control plasma were screened during the method validation, and no interference was observed (Fig.8and9) shows, the extracted 5 ng/ml standard and the extracted 15 ng/ml QC , respectively. In each chromatogram, the I.S concentration was 50 ng/ml. The chromatogram are sharp and with baseline resolution.

Fig.7.Chromatogram of Blank along with I.S

Fig.8. Chromatogram of 5ng/ml

Fig.9. Chromatogram of 15 ng/ml

Method validation:

Specificity and Selectivity:

No interference was observed in six different lots of drug free human plasma samples used for analysis, at the retention time of either analyte or internal standard. It was shown in table 1.

Table.1. Specificity and Selectivity

S. No	Lot No.	Description	Blank		Average	Blank Spiked with LLOQ		Average	% interference
S. No	Lot No.	Description	1	2	Average	1	2	Average	% interference
1	1	Analyte Area	4.958	18.569	11.7635	178.601	171.806	175.2035	6.7
2	1	IS Area	18.993	21.795	20.3940	18156.559	15982.700	17069.6295	0.1
3	2	Analyte Area	0.000	6.454	3.2270	143.519	152.069	147.7940	2.2
4	2	IS Area	0.000	1.391	0.6955	15234.881	15712.300	15473.5905	0.0
5	3	Analyte Area	0.565	1.437	1.0010	166.453	168.564	167.5085	0.6
6	3	IS Area	0.847	1.333	1.0900	16261.716	16439.975	16350.8455	0.0
7	4	Analyte Area	0.672	0.427	0.5495	184.925	154.316	169.6205	0.3
8	4	IS Area	1.196	0.911	1.0535	16875.475	15850.722	16363.0985	0.0
9	5	Analyte Area	1.066	0.000	0.5330	187.485	178.507	182.9960	0.3
10	5	IS Area	2.733	0.840	1.7865	18654.354	17714.549	18184.4515	0.0
11	6	Analyte Area	1.408	2.886	2.1470	151.791	164.129	157.9600	1.4
12	6	IS Area	1.465	0.891	1.1780	16090.925	16419.871	16255.3980	0.0

Table.2. Matrix Effect of Esomeprazole in Human plasma

Description		Un Extracted Samples			Neat solution Samples
S. No	Replicate	LQC	MQC	HQC	LQC	MQC	HQC
1	1	437.449	3045.81	21201.438	501.462	3640.359	21433.604
2	2	389.22	3312.963	22796.119	498.936	3666.873	17191.781
3	3	473.967	3568.591	20733.768	469.782	3706.368	19065.26
4	4	498.814	3048.888	21249.457	501.333	3664.179	20248.375
5	5	461.542	3253.739	21355.398	479.811	3350.474	20977.914
6	6	451.162	3313.685	21428.465	459.193	3672.014	21486.717
Average		452.0257	3257.2793	21460.770	485.0862	3616.7112	20067.2752
Standard Deviation		37.19921	195.63026	697.9602	18.20135	132.14205	1674.28976
% Coefficient of Variance		8.2	6.0	3.25	3.8	3.7	8.3
% Matrix Effect		-6.8	-9.9	10.3
Average % Matrix Effect		-2.1

Table.3. Back Calculated Concentrations (ng/mL) of Esomeprazole Calibration Standards in Human plasma

S. No	Description	Replicate	STD 1 5.000 ng/ml	STD 2 10.000 ng/ml	STD 3 25.000 ng/ml	STD 4 85.000 ng/ml	STD 5 200.000 ng/ml	STD 6 500.000 ng/ml	STD 7 850.000 ng/ml	STD 8 1000.000 ng/ml
1	P And A 01	1	4.766	8.955	28.468	79.577	208.885	496.002	793.813	1036.062
2	P And A 01	2	5.662	8.881	24.652	76.060	198.667	503.377	858.004	1044.613
3	P And A 02	1	4.931	9.089	27.679	79.467	195.128	488.726	867.004	1019.425
4	P And A 02	2	5.285	9.134	28.539	76.972	196.570	495.096	842.478	973.530
5	P And A 03	1	4.068	11.188	26.949	87.975	198.800	425.405	878.816	1053.395
6	P And A 03	2	3.548	9.794	22.495	89.950	212.260	435.552	813.059	1065.294
Average			4.7100	9.5068	26.4637	81.6668	201.7183	474.0263	842.1957	1032.0532
Standard Deviation			0.78119	0.88528	2.41120	5.84959	7.07464	34.20149	32.85213	32.61496
% Coefficient of Variance			16.6	9.3	9.1	7.2	3.5	7.2	3.9	3.2
% Difference From Nominal			-5.8	-4.9	5.9	2.1	0.9	-5.2	-0.9	3.2

Table.4. Intra Batch Back Calculated Concentrations (ng/mL) of Esomeprazole Quality Control Samples in Human plasma

Description		P And A 01				P And A 02				P And A 03
S. No	Repli cate	LLOQ 5.000 ng/ml	LQC 15.000 ng/ml	MQC 110.000 ng/ml	HQC 800.000 ng/ml	LLOQ 5.000 ng/ml	LQC 15.000 ng/ml	MQC 110.000 ng/ml	HQC 800.000 ng/ml	LLOQ 5.000 ng/ml	LQC 15.000 ng/ml	MQC 110.000 ng/ml	HQC 800.000 ng/ml
1	1	5.071	17.359	116.087	845.247	5.602	17.137	118.961	811.897	3.595	15.118	108.427	763.737
2	2	4.348	17.232	120.258	864.065	4.965	17.045	114.777	833.481	3.530	19.540	109.855	777.603
3	3	4.366	18.015	118.217	767.131	5.122	17.310	115.512	823.069	3.764	14.211	119.964	841.338
4	4	5.012	17.447	120.059	784.211	5.262	16.980	111.307	844.915	4.443	16.085	109.519	784.301
5	5	4.925	16.592	121.570	832.887	5.421	16.122	113.027	835.211	4.664	16.833	102.353	701.727
6	6	5.328	15.186	122.701	840.656	5.510	17.174	109.362	795.126	4.155	13.987	101.631	805.407
Average		4.8417	16.9718	119.8153	822.3662	5.3137	16.9613	113.8243	823.9498	4.0252	15.9623	108.6248	779.0188
Standard Deviation		0.39876	0.98641	2.37135	37.98492	0.24256	0.42649	3.37717	18.07792	0.46861	2.06179	6.62085	46.51340
% Coefficient of Variance		8.2	5.8	2.0	4.6	4.6	2.5	3.0	2.2	11.6	12.9	6.1	6.0
% Difference From Nominal		-3.2	13.1	8.9	2.8	6.3	13.1	3.5	3.0	-19.5	6.4	-1.3	-2.6

Table.5. Inter Batch Back Calculated Concentrations (ng/mL) of Esomeprazole Quality Control Samples in Human plasma

S. No	Description	Replicate	LLOQ 5.000 ng/ml	LQC 15.000 ng/ml	MQC 110.000 ng/ml	HQC 800.000 ng/ml
1	P And A 01	1	5.071	17.359	116.087	845.247
2		2	4.348	17.232	120.258	864.065
3		3	4.366	18.015	118.217	767.131
4		4	5.012	17.447	120.059	784.211
5		5	4.925	16.592	121.570	832.887
6		6	5.328	15.186	122.701	840.656
7	P And A 02	1	5.602	17.137	118.961	811.897
8		2	4.965	17.045	114.777	833.481
9		3	5.122	17.310	115.512	823.069
10		4	5.262	16.980	111.307	844.915
11		5	5.421	16.122	113.027	835.211
12		6	5.510	17.174	109.362	795.126
13	P And A 03	1	3.595	15.118	108.427	763.737
14		2	3.530	19.540	109.855	777.603
15		3	3.764	14.211	119.964	841.338
16		4	4.443	16.085	109.519	784.301
17		5	4.664	16.833	102.353	701.727
18		6	4.155	13.987	101.631	805.407
Average			4.7268	16.6318	114.0882	808.4449
Standard Deviation			0.65469	1.35177	6.32748	40.19540
% Coefficient of Variance			13.9	8.1	5.5	5.0
% Difference From Nominal			-5.5	10.9	3.7	1.1

Table.6. Recovery of Esomeprazole in Human plasma

Description		Extracted Samples			Un extracted Samples
S. No	Replicate	LQC	MQC	HQC	LQC	MQC	HQC
1	1	308.283	2446.995	14582.405	437.449	3045.810	25201.438
2	2	334.492	2197.919	16334.256	389.220	3312.963	22796.119
3	3	372.892	2583.599	17405.057	473.967	3568.591	20733.768
4	4	358.230	2483.083	14382.025	498.814	3048.888	23249.457
5	5	371.572	2771.524	14341.440	461.542	3253.739	25355.398
6	6	374.168	2316.820	14784.331	451.162	3313.685	21428.465
Average		353.2728	2466.6567	15304.9190	452.0257	3257.2793	23127.4408
Standard Deviation		26.67492	200.97113	1268.31997	37.19921	195.63026	1897.68849
% Coefficient of Variance		7.6	8.1	8.3	8.2	6.0	8.2
% Recovery		78.2	75.7	66.2
Average % Recovery		73.4

Table.7. Recovery of Rabeprazole (IS) in Human plasma

Description		Extracted Samples			Un extracted Samples
S. No	Replicate	LQC	MQC	HQC	LQC	MQC	HQC
1	1	10789.813	11768.158	10718.741	14242.957	14617.326	16013.591
2	2	11516.692	11094.775	11161.039	14088.903	15578.989	14749.769
3	3	12728.305	12606.742	11679.031	15556.506	15805.459	14910.367
4	4	11688.553	11648.104	10400.96	15406.089	14919.652	15514.557
5	5	12256.018	12291.632	11001.625	14726.53	15085.920	15910.995
6	6	12280.102	11457.379	10419.361	15234.936	15202.416	14937.318
Average		11876.5805	11811.1317	10896.7928	14875.9868	15201.6270	15339.4328
Standard Deviation		689.66292	553.28274	489.53165	618.95464	433.89433	548.69632
% Coefficient of Variance		5.8	4.7	4.5	4.2	2.9	3.6
% Recovery		79.8	77.7	71.0
Average % Recovery		76.2

Matrix Effect:

Matrix effect was determine by comparing the LC-MS/MS response of Esomeprazole at a concentration of 15 ng/ml, 110 ng/ml and 800 ng/ml to the LC-MS/MS response of the analyte present in reconstitution solution. The matrix effect of Esomaprazole at a concentration of 15 ng/ml, 110 ng/ml and 800 ng/ml was -6.8%, -9.9%, 10.3% respectively. The mean absolute matrix effect was -2.1%, indicates responses in the reconstitution solution and plasma extract are same and no absolute matrix effect and no ion suppression was observed. It was shown in table 2.

Precision and Accuracy:

Data for intra batch and inter batch permissible and accuracy of the method for Esomeprazole are present in Table 3,4and5. the accuracy deviation values are within 15% (Except 5ng/ml was 20%) of the actual values. The precision determined at each concentration level does not exceed 15% (Except 5 ng/ml was 20%) of the relative standard deviation (RSD) the results revealed good precision and accuracy.

Recovery:

Percentage recovery of Esomeprazole was measured by dividing the ratio of concentration levels with that of controls. Then mean recoveries for Esomeprazole (15,110 and 800 ng/ml) were 78.2%, 75.7% and 66.2% respectively. The mean recovery of internal standard Esomeprazole was 76.2%. It was shown in table 6and7.

Freeze thaw stability:

The freeze thaw stability of Esomeprazole was determined by measuring the accuracy and precision for samples that underwent three freeze thaw cycles. The results showed Esomeprazole was stable in human plasma through three freeze thaw cycles. The precision of the QC samples LQC (15 ng/ml) and HQC 800 ng/ml) were 4.6 and 4.8 respectively. The accuracy of the QC samples LQC (15 ng/ml) and HQC (800 ng/ml) were 12.3 and 4.3 respectively for Esomeprazole. It was shown in table 8.

Table.8. Freeze Thaw Stability of Esomeprazole in Human plasma

S. No	Replicate	LQC 15.000 ng/ml	HQC 800.000 ng/ml
1	1	17.040	773.911
2	2	17.293	848.150
3	3	16.745	871.962
4	4	15.837	879.818
5	5	16.164	806.037
6	6	17.993	827.320
Average		16.8453	834.5330
Standard Deviation		0.78057	40.43800
% Coefficient of Variance		4.6	4.8
% Difference From Nominal		12.3	4.3

Number of FT Cycles = 3

Bench Top Stability:

At ambient temperature (20-30⁰C) Esomeprazole was found to be stable for 6 hrs in human plasma. The precision observed were QC samples LQC (15 ng/ml) and (HQC 800 ng/ml) were 5.2 and 4.6 respectively. The accuracy of QC sample LQC (15 ng/ml) and HQC (800 ng/ml) were 9.1 and 3.3 respectively, It was shown in table 9.

Table.9. Bench Top Stability of Esomeprazole Quality Control Samples in Human plasma

S. No	Replicate	LQC 15.000 ng/ml	HQC 800.000 ng/ml
1	1	16.395	800.927
2	2	16.967	861.610
3	3	15.004	877.377
4	4	16.206	814.059
5	5	17.088	767.429
6	6	16.531	835.358
Average		16.3652	826.1267
Standard Deviation		0.85524	38.25798
% Coefficient of Variance		5.2	4.6
% Difference From Nominal		9.1	3.3

Duration of Bench Top Exposure = 6 hr

Auto injector stability:

Stability of samples stored in auto injector was carried out over a period of 68 hrs by injecting the QC sample. The accuracy observed were QC sample LQC (15 ng/ml) MQC (110 ng/ml)and HQC ( 800 ng/ml) were 11.8,-1.2 and 3.3 respectively. It was shown in table 10.

Table.10. Auto injector Stability of Esomeprazole in Reconstitution Solvent

S. No	Replicate	LQC 15.000 ng/ml	MQC 110.000 ng/ml	HQC 800.000 ng/ml
1	1	17.441	103.545	831.508
2	2	16.111	113.773	821.206
Average		16.776	108.659	826.357
% Difference From Nominal		11.8	-1.2	3.3

Duration = 68 hrs

Long term stock solution stability:

The working solutions of Esomeprazole were found to be stable for 23 days at -20^oC. These data showed in the table11.

Table.11. Long Term Stability of Esomeprazole in Methanol after 23 days at -20 °C

S. No	Replicate	Response Ratio
S. No	Replicate	Stability Sample	Comparison Sample
1	1	0.858	0.876
2	2	0.8603	0.7884
3	3	0.8242	0.7882
4	4	0.8333	0.9143
5	5	0.8405	0.8492
6	6	0.7893	0.7996
Average		0.8343	0.8359
Standard Deviation		0.02607	0.05247
% Coefficient of Variance		3.1	6.3
% Stability		-0.2

Long term frozen stability:

Esomeprazole was stable at -70⁰C for 29 days in human plasma. The accuracy for the LQC (15 ng/ml) and HQC (800 ng/ml) samples was 3.6 and 0.7 over the stability testing period in deep freezer at -70⁰C. It was shown in table 12.

Table.12. Long Term Frozen Stability of Esomeprazole Quality Control

S. No	Replicate	LQC 15.000 ng/ml	HQC 800.000 ng/ml
1	1	14.808	758.939
2	2	16.050	845.275
3	3	16.215	846.53
4	4	15.667	771.024
5	5	15.248	778.479
6	6	15.247	835.316
Average		15.5392	805.9272
Standard Deviation		0.53643	40.59573
% Coefficient of Variance		3.5	5.0
% Difference From Nominal		3.6	0.7

Samples in Human plasma Stored at -70 °C

CONCLUSION:

The bioanalytical methodology described in this manuscript was specific, sensitive, accurate and precise. The method employed HPLC coupled with electrospray ionization mass spectrometric detection (LC-ESI-MS). The method involved a simple sample preparation by liquid- liquid extraction followed by isocratic chromatographic separations. The LC-ESI-MS method was capable of estimating 5 ng/ml of Esomeprazole accurately in human plasma with high degree of reproducibility.

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Received on 10.12.2009 Modified on 03.02.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 477-484