Development and Validation of High Performance Thin Layer Chromatography for Determination of Esomeprazole Magnesium in Human Plasmaof a Method for Regioisomer Impurity Detection and Quantitation within the Raw Material 3-Chloro-5-Fluorophenol by Gas Chromatography

A simple, sensitive, rapid and economic high performance thin layer chromatographic method has been developed for determination of Esomeprazole magnesium in human plasma by liquid-liquid extraction. The plasma sample was extracted using chloroform. A concentration range from 200-700 ng/spot of Esomeprazole magnesium was used for calibration curve. The percent recovery of Esomeprazole magnesium was found 101.61 percent. The mobile phase constitute of ethyl acetate: methanol: ammonia (32%) (9:1: 0.5 v/v). Densiometric analysis was carried out at wavelength 301 nm. The Rf value for Esomeprazole magnesium was found 0.54 ± 0.05. The stability of Esomeprazole magnesium in human plasma was confirmed during freeze thaw cycles at -30°C, on bench top during 24 h at room temperature and post preparative for 48 h. The proposed method was validated statistically by performing recovery study for determination of Esomeprazole magnesium in human plasma by liquid-liquid extraction.


Introduction
Esomeprazole magnesium dihydrate1 (ESO), bis(5-methoxy-2-[(S)-[(4-methoxy-3,5dimethyl-2-pyridinyl) methyl] sulfinyl]-1-Hbenzimidazole-1-yl) magnesium dihydrate (Figure 1) a, is a compound that inhibits gastric acid secretion. ESO is cost-effective in the treatment of gastric oesophageal reflux diseases. ESO is the S-isomer of omeprazole, the first single optical isomer proton pump inhibitor, generally provides better acid control than current racemic proton pump inhibitors and has a favorable pharmacokinetic profile relative to omeprazole. Several methods have been employed for the estimation of ESO alone and combination with other drugs such as UV and RP-HPLC methods. Literature survey reveals that many analytical methods such as UV spectrophotometric [1][2][3][4], HPLC methods [5][6][7][8][9][10], LCMS [11], HPTLC [12][13][14] methods are reported for determination of Esomeprazole magnesium individually as well in combination. Specific and sensitive methods based on mass spectrometry methods were reported earlier. Earlier reports on HPLC based bioanalytical estimation of esomeprazole resulted in lesser sensitivity, and high noise in the base line indicating a need to develop a more efficient, sensitive, simple and rapid method in human plasma. To access the reproducibility and wide applicability of the developed method, it was validated as per FDA guidelines [15].

Materials and Methods
Instrumentation HPTLC Camag with precoated silica gel Plate 60F254 (20 cm × 10 cm) 250 µm thicknesses (E. Merck, Darmstadt, Germany) was used as stationary phase. Sample application was done by using Camag 100 µl syringe and Camag Linomat V applicator. The sample was sprayed in the form of narrow bands of 8 mm length at a constant rate 2 µl/s. Linear ascending development was carried out in 20 cm × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland). The densitometric scanning was performed by using Camag TLC scanner III supported by win CATS software (V1.4.2.8121 Camag). Evaluation of chromatogram was done by using peak areas of drug.

Chemicals
Esomeprazole magnesium (A.S Bulk drugs, Hyderabad, India), was used as such by without checking their purity. The HPLC grade methanol and Analytical Reagent grade ethyl acetate, ammonia solution, chloroform was purchased from Modern sciences, Nashik, India. Human plasma used for research work was supplied by Arpan Blood Bank, Nashik, Maharashtra, India.

Chromatographic condition
Mobile phase was selected as mixture of Ethyl acetate: methanol: ammonia solution (32%) in the ratio of (9:1:0.5 v/v/v) for the development of plates. Time for chamber saturation was optimized to 20 min. The length of chromatographic development was 70 mm. The densitometric scanning was performed at 301 nm.

Preparation of stock solution and working standard solution
Stock solutions1 mg/ml each of Esomeprazole magnesium was prepared in methanol.

Preparation of plasma sample
To 1 ml of fresh human plasma in separate 15 ml of centrifuge tubes, spiked 1 ml of drug solution from 1000 mg/ml stock solution. Wait for C max i.e up to T max of drug (1 and ½ h) to reach equilibrium concentration. 5 ml extracting solvent chloroform was then added in stock to the test solution prepared from tablet formulation at three different spiking level 80%, 100%, 120% of target concentration.

Recovery:
Recovery experiments was performed by comparing the analytical result for extracted samples at three concentrations (200, 400, 600 ng/spot) with unextracted standards that represent 100% recovery.

Limit of detection (LOD) and limit of quantitation (LOQ):
The parameters LOD and LOQ were determined using the signal-tonoise ratio by comparing results of the test of samples with known concentrations of analyte to blank samples. The analyte concentration that produced a signal-to noise ratio of 3:1 was accepted as the LOD. The LOQ was identified as the lowest plasma concentration of the standard curve that could be quantified with acceptable accuracy, precision and variability.

Stability
Bench top stability: A Stock solution of Esomeprazole magnesium was kept at room temperature for 24 hours.
Post preparative stability: A Stock solution of Esomeprazole magnesium was kept at room temperature for 48 days.
Freeze thaw stability: The stability of low and high quality control samples were determined after three freeze thaw cycles.

Results and Discussion
Extraction Procedure Optimization One of the most difficult task during the method development was to achieve a high and reproducible recovery from the solvent, which is used for extraction of the drug centrifuge tube. Tube was cycomix for 5 min and then centrifuged at 3000 for 20 min. After centrifugation process, the supernatant liquid was collected in another tube and volume make up to 10 ml with methanol. The analysis was carried on HPTLC.

Method validation
Calibration plot: The calibration plot for the HPTLC method was constructed by analysis of six solutions containing different concentrations of Esomeprazole magnesium (200-700 ng/ml). The Esomeprazole magnesium can be determined at LLOQ 2 µl/ml. In the range 200-700 ng/ml the data were best fitted by a linear equation y=mx+b, the coefficient of determination (R2) was 0.9958.

Selectivity:
For selectivity, analyses of blank sample of the appropriate biological matrix (plasma) were done. Each blank sample was tested for interference, and selectivity was ensured at the lower limit of quantification (LLOQ) i.e., 200 ng/ml [16,17].
Precision: Precision was measured using a minimum of three determinations per concentration (200, 400, 600 ng/spot). Intraday precision (Repeatability) was performed by taking three different concentrations (200, 00, 600 ng/spot) covering specified range in the triplicates and were analyzed three times within a day with same operator and with same equipment. Inter day precision was determined by analyzing three different concentrations (200, 400, 600 ng/spot) in triplicates on three different days within same laboratory conditions. Accuracy: Accuracy was determined by replicate analysis of samples containing known amounts of the analyte (200, 400, 600 ng/ spot). The study was determined by spiking known amount of standard  (Table 1). Different solvents were tried for the extraction of from human plasma. Densitogram obtained shows the Rf at 0.52 and is shown in Figure 1. HPTLC densitogram of 200-700 ng/band was analyzed and observed 3-D view was shown in Figure 2.

Linearity
The linear plot was observed in the concentration range of 200-700 ng/spot. Results obtained are shown in Table 2 and calibration plot obtained was shown in Figure 2.

Precision
Intraday and interday precision of Esomeprazole magnesium in human plasma assures the repeatability of test results. The % RSD found was below 2. Results of intraday and interday precision were shown in Table 3 and Table 4, respectively.

Accuracy
Accuracy was studied by standard addition method and % recovery found was within acceptable limit. Results of recovery study are shown in Table 2 and Table 4 and statistical validation is shown in Table 5.

Selectivity
Analyses of blank sample of the appropriate biological matrix (plasma) were done. Each blank sample was tested for interference,

Sn no
Solvents used Recovery          Table 9: Results for post preparative stability of Esomeprazole in human plasma.