INTRODUCTION:

Esomeprazole magnesium chemically describes (S)-5-methoxy-2- [(4-methoxy-3, 5-dimethylpyridin-2-yl) methylsulfinyl]-3H-benzoimidazole magnesium salt is an antiulcervative agent¹, and which is used in the treatment of dyspepsia, peptic ulcer disease (PUD), gastroesophageal reflux disease (GORD/GERD) and Zollinger-Ellison syndrome.

In the synthesis of Esomeprazole magnesium (1), p-Anisidine (2) and 4-Methoxy-2-nitro aniline (3) are important reagents. The chemical structures of the compounds are given in Scheme 1. Identification and determination of these two impurities in 1 is essential, because of their genotoxic nature². As per the regulatory guidelines³, a threshold of toxicological concern (TTC) value of 1.5 µg day^-1 in take of a toxic impurity is permitted. The permitted quantity in ppm is the ratio of TTC in microgram day^-1 and dose in gram day^-1. Since, 40 mg of Esomeprazole magnesium is administered per day⁴ in the form of tablets (20 mg to 40 mg with the trade name Nexium), the estimated permissible quantity of these impurities is 37.5 ppm per day.

High-performance liquid chromatography (HPLC) is well established and robust but it has low sensitivity, requires large amounts of solvents and time consuming process. An approach based on gas chromatography (GC)-mass spectrometry (MS) is feasible within the limits of time and expensive.

Despite the importance of the issue no analytical method has been proposed on the quantitation of genotoxic impurities (2, 3, 4) present in Esomeprazole magnesium (1). Hence, a selective and sensitive GC-MS method was developed for the identification and determination of these impurities in Esomeprazole.

EXPERIMENTAL:

Chemicals:

Esomeprazole magnesium (1), p-Anisidine (2), and 4-Methoxy-2-nitroaniline (3) were received from the process research department of Matrix laboratories ltd., India. HPLC-grade methanol was purchased from Merck chemicals (Mumbai, India).

Preparation of standard solutions:

The stock solutions of p-Anisidine (2) and 4-Methoxy-2-nitro aniline (3) were prepared by dissolving about 10.0 mg each (individually in separate 100 mL volumetric flasks) of the compounds in methanol. The diluted stock solution was prepared by pippeting 0.4 mL of the p-Anisidine (2) and 1.0 mL of 4-Methoxy-2-nitroaniline (3) into a 100 mL volumetric flask and diluting to volume with the methanol. The working standard solution of p-Anisidine (2) is 1.05 ppm and 4-Methoxy-2-nitro aniline (3) is 3.16 ppm was prepared by further diluting 3.0 mL of the diluted stock solution into 10 mL volumetric flask. The sample solution was prepared by accurately weighing about 100 mg of the Esomeprazole magnesium drug substance into a 2 mL GC vial and adding 1.0 mL of the methanol. The concentration of the p-Anisidine (2) is 1 ppm and 4-Methoxy-2-nitro aniline (3) is 3.16 ppm with respect to 100 mg mL^-1 of Esomeprazole magnesium.

Scheme 1. Structures of the studied compounds.

Instrumentation:

An Agilent 7890A GC system coupled with 5975C quadrupole mass spectrometer and auto sampler (Agilent Technologies, Model No. 7683B) was used in the electron ionization (EI) mode (Agilent Technologies, PA, USA). Data acquisition and processing were conducted using the Turbo mass software (MSD Chem station E.02.00) on a Pentium computer (Digital equipment Co.). Agilent liner (Agilent Technologies, part no. 5183-4647) was used in GC and the empty liner was washed by keeping sonication in chloroform/water/methanol solvents differently followed by drying for 10 minutes then reused with new wool.

Operating conditions:

The analytes were separated using JandW Scientific DB-5 capillary column (30 m x 0.53 mm I.D. x 5.0 µm). High purity helium (99.999%) was used as the carrier gas at a constant flow rate of 1.0 mL min^-1. One micro litre of sample was injected in the split less mode. The injector and GC-MS interface temperatures were set at 180 °C and 230 °C, respectively, and the oven temperature program was as follows: initial temperature 80 °C for 5 min, increased to 220 °C at 10 °C min^-1 and maintained at this temperature for 10 min; the total GC run time, 29 min. The mass spectrometer was operated in the electron ionization mode at 70 eV and the ion source temperature was set at 230 °C. The samples were injected with the Agilent auto sampler.

The quantitative analysis was carried out in the selected ion monitoring (SIM) or selected ion-recording (SIR) mode. The mass spectra of 2 and 3 gave molecular ions of m/z 123 and m/z 168, respectively. The corresponding base peaks were at m/z 108 and m/z 168 in each case. Thus, m/z 108 was chosen for 2, and molecular ion m/z 168 for 3 were chosen to monitor SIM experiments.

RESULTS AND DISCUSSION:

Method development and optimization:

The challenge was to achieve the desired detection and qauntitaion at very low levels using gas chromatography-mass spectrometry (GC-MS). To obtain good separation and the desired sensitivity, one approach is to select either most prominent fragment ion as selective ion recording mode (SIR) in MS and if require increase the sample amount injected into the GC-MS system. To decrease the interference of other substances with 2 and 3 other fragments also can be selected as selective ion recording (SIR) mode and suitable gradient column temperature in combination with a moderate inlet temperature (200 ⁰C) may allow a large injection volume without significant deterioration in column efficiency.

The effect of concentration on separation and quantitation of 2 and 3 were investigated by injecting 1 µL of the stock solution and working standard solutions of 1.0 ppm and 3.16 ppm, respectively. Further studies were not done to determine the maximum injection.

Method validation

The validation work was conducted according to the ICH (International Conference on Harmonization) guidelines [5-8]. The validated method parameters include specificity, limits of detection (LODs), and limit of quantitaion (LOQs), precision, linearity and accuracy.

The specificity for 2 and 3 were showed in a total ion chromatogram (Fig. 1). The detection limit (LOD) of the method for 2 and 3 was estimated from a SIR chromatogram of a solution containing about 0.4 ppm for 2 and 1.23 ppm for 3. From the selective ion recording chromatograms a signal to noice ratio of 2.3 and 3.4, was obtained for 2 and 3, respectively. A second instrument (Same manufacturer) was used to repeat the experiments and similar results were obtained. Esomeprazole magnesium at 100 mg mL^-1 was spiked with 1.05 ppm and 3.16 ppm for 2 and 3. All three peaks have a signal to noice ratio of near about 3, indicating that this method is capable of detecting about 0.4 ppm level of 2 and 1.0 ppm of 3 in the drug substance of 1.

Figure 1. Total ion chromatogram of p-Anisidine (2) (6. 201 min.) and 4-Methoxy-2-nitroaniline (3) (16.465 min.).

In the pharmaceutical industry, the quantitation limit (LOQ) was defined as the lowest amount of analyte in a sample that can be quantitatively determined with suitable precision and accuracy. The LOQ was determined less than or equal to 1.05 µg g^-1 (1.05 ppm) and 3.16 µg g^-1 (3.16 ppm) for 2 and 3.

Linearity of the method was determined by preparing and analyzing a series of 5 standard solutions to cover the concentration range of LOQ to 3.3 ppm for 2 and 8.5 ppm for 3. Regression analysis of the peak area versus concentration data yields an R²> 0.98.

The experimental results also show that this method has excellent precision without using an internal standard. Multiple injections were made for the standard solutions containing 1.05 ppm and 3.16 ppm each of 2 and 3. For six injections of the standard solutions, the percentage of R.S.D of the peak area was 3.55 and 8.59 (Table 1).

Table 1. Analytical data of proposed methods.

Parameters

p-Anisidine

(2)

4-Methoxy-2-nitroaniline (3)

LOD (ppm)^a

LOQ (ppm)^a

Linear range (ppm)^a

Slope

Intercept

Correlation coefficient

Repeatability (% RSD)^b

Accuracy

0.4

1.05

1-3

931.36

216.49

0.9920

3.55

97.1 - 99.3

1.05

3.16

3-8

179.46

955.26

0.9923

8.59

97.6 - 104.7

^a LODand LOQ values are given with respect to 50 mg mL^-1.

^b Six determinations at LOQ.

Accuracy of the method was determined by analyzing a drug substance samples spiked with known amount of the 2 and 3. The spiked levels were 1.05 and 3.16 ppm. The recovery is in the range of 85.9 to 100.3, respectively. Because this method uses the dissolve-and-inject approach, for every simple injection, about 1 µL (100 mg mL^-1) of the drug substance is introduced into the injection port. The accumulation of drug substance may have negative effect on the recovery. Therefore the injection liner should be washed after every sequence of 10-15 injections.

CONCLUSIONS:

A simple and sensitive gas chromatography-mass spectrometric (GC-MS) method has been developed and validated for the trace analysis of p-Anisidine (2) and 4-Methoxy-2-nitro aniline (3) in Esomeprazole magnesium (1) drug substances. Under the optimal conditions, the recovery for p-Anisidine is between 85.9% and 90.0% and 4-Methoxy-2-nitroaniline is between 99.2% to 100.3% spiked samples at the three levels concentration in sample preparation. Limits of quantification (LOQ) and limits of detection (LOD) of the standard solution for p-Anisidine is 0.001 and 0.0004 µg g^-1 and for 4-Methoxy-2-nitroaniline is 0.003 and 0.001 µg g^-1, respectively. Linear range from 0.001 to 0.003 µg g^-1 is obtained with correlation coefficient of 0.9920 for p-Anisidine and Linear range from 0.003 to 0.008 µg g^-1 is obtained with correlation coefficient of 0.9923 for 4-Methoxy-2-nitro aniline. From these results it can be concluded that this GC-MS method can be conveniently applied to simultaneously determination of p-Anisidine (2) and 4-Methoxy-2-nitro aniline (3), which are present in Esomeprazole magnesium in the lowest level of detection limits.

ACKNOWLEDGEMENTS:

The authors thank the management of Matrix Laboratories Ltd. for supporting this work. Authors wish to acknowledge the Process Research Department for providing the samples for this research. We also would like to thank colleagues from the separation science division of Analytical Development for their co-operation in carrying out this work.

REFERENCES:

1. The Merck Index (Merck andCo., Inc white house station, NJ, USA (2006) 14 th edn. pp 1179.

2. Elder DP, Teasdale A, and Lipczynski AM. Control and analysis of alkyl and benzyl halides and other related reactive organohalides as potential genotoxic impurities in active pharmaceutical ingredients (APIs). J Pharm Biomed Anal 2008; 46:1.

3. European medicines Agency guideline on the limits of genotoxic impurities, CPMP/SWP/5199/02 EMEA/CHMP/QWP/b 251344/2006

4. Thomson PDR, Montvale NJ (2007) physician’s desk.

5. ICH Q2A: Text on validation of analytical procedures: terms and definitions, International Conference on Harmonization, Fed, Reg. (60 FR 11260), 1 March 1995.

6. ICH Q2B: Text on validation of analytical procedures: methodology, In: International Conference on Harmonization, Fed, Reg. (62 FR 27463), 19 May 1997

7. ICH Q3A: Impurities in new drug substances, In: International Conference on Harmonization, Fed, Reg. (68 FR 6924), 11 February 2003.

8. ICH Q3B: Impurities in new drug products, In: International Conference on Harmonization, Fed, Reg. (62 FR 27454), 19 May 1997.

Received on 09.01.2010 Modified on 27.02.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 395-397