Advanced Analytical Methods: LC-MS/MS with Multiple Reaction Monitoring (MRM Mode) For the Quantification of Two Potential Genotoxic Impurities in Emtricitabine Drug Substance

Received: 20-Dec-2023, Manuscript No. DPC-23-123113; Editor assigned: 23-Dec-2023, Pre QC No. DPC-23-123113; Reviewed: 08-Jan-2024, QC No. DPC-23-123113; Revised: 21-Feb-2025, Manuscript No. DPC-23-123113; Published: 28-Feb-2025, DOI: 10.4172/0975-413X.17.2.638-643

Introduction

A nucleoside reverse transcriptase inhibitor called emtricitabine is used to treat HIV (human immunodeficiency virus) infection in adults. The medication copies HIV RNA (ribonucleic acid) into new virus DNA (deoxyribonucleic acid) by suppressing the reverse transcriptase enzyme. Tenofovir disoproxil fumarate is frequently given in conjunction with emtricitabine; the daily maximum dose of emtricitabine is 0.2 g. Because they are reactive by nature, synthetic starting materials and intermediates could appear as contaminants in the finished API. These substances are frequently carcinogens or mutagens due to the biological reactivity that commonly results from the chemical reactivity [1-4]. It has been repeatedly demonstrated that the significant chemical reactivities of the various alkylating agents prevented their retention within the final API (Figure 1).

Figure 1: Structure of emtricitabine.

L-methyl glyoxylate monohydrate (LMGH) is a crucial step in the production of lamivudine and emtricitabine 1, two significant antiretroviral medications. These two commonly prescribed medications serve as cytidine reverse transcriptase inhibitors in combination therapy for the treatment of HIV-1, HIV-2, and Hepatitis B. Since Sub-Saharan Africa has a higher rate of AIDS cases than any other region in the world, these generic medications are especially crucial in these nations (Figure 2).

Figure 2: Structure of L-menthyl glyoxylate monohydrate.

2-methoxy 4-amino-5-fluro pyramidine introduction part (Figure 3).

Figure 3: Structure of 2-methoxy 4-amino-5-fluro pyramidine.

Based on the maximum daily dosage of Emtricitabine L -Menthyl glyoxylate monohydrate (LMGH) and 2-Methoxy 4-Amino-5-Fluro pyramidine (MAFP) are required to be controlled at a limit of NMT 6.25 µg/g and NMT 6.25 µg/g respectively in the API.

L-Menthyl glyoxylate monohydrate and 2-Methoxy 4-Amino-5-Fluro pyramidine are used in the synthesis of Emtricitabine.Route of synthesis shown below (Figure 4).

Figure 4: L-Menthyl glyoxylate monohydrate and 2-Methoxy 4-Amino-5-Fluro pyramidine.

Materials and Methods

The variety of analytical procedures has expanded and there has been a general revival due to the growing regulatory awareness on the potential risks. For the determination of L-Menthyl glyoxylate monohydrate (LMGH) and 2-Methoxy 4-Amino-5-Fluro pyramidine (MAFP), the suggested LC-MS/MS (liquid chromatography/mass spectrometery/mass spectrometery) approach provides a straightforward, robust, and labor-saving alternative that requires no time-consuming sample preparation steps. When comparing this method to arduous sample preparation techniques, it has numerous advantages over the method documented in the literature in terms of specificity, accuracy, and reproducibility involving direct analysis [5-7]. In order to deal with matrix interference, a diluent that makes the emtricitabine API insoluble and allows for the extraction of analytes from the API matrix is used. The suggested technique uses electrospray ionization in the MRM mode to quantify LMenthyl glyoxylate monohydrate (LMGH) and 2-Methoxy 4-Amino-5-Fluro pyramidine (MAFP).

Experimental

Chemical and Reagents

List of chemicals, reagents, chemicals, sample, standards and impurities show in Table 1.

Table 1: List of Chemicals and Reagents.

Instrumentation

For L -Menthyl glyoxylate monohydrate (LMHG) and 2-Methoxy 4-Amino-5-Fluro pyramidine (MAFP): An ultra-performance liquid chromatography, acquity H-class system, with a quaternary solvent manager, gradient mixer assembly, sample manager-F.T.N (Flow through needle) auto injector, with a column oven coupled to Xevo TQ-S Triple Quadrupole LC/MS/MS Mass Spectrometer, (Make Waters).Column was employed in the method was acquity UPLC CSH Phenyl Hexyl 1.7 µm. (150 mm × 2.1 mm) and X-Select CSH C18 150 × 4.6 3.5 µm mm respectively. All the weighing in the experiments was done with Sartorius balance capable of measuring with an accuracy of 0.01 mg.

Chromatographic conditions for LC

Table 2: Chromatographic Conditions for LC.

Mass spectroscopic conditions

Source type: ESI

Mode of ionization: Positive

Source parameters

Capillary: 3.9 kv

Source temperature: 150°C

Desolvation temperature: 500°C

Cone gas flow: 150 L/Hr

Desolvation gas flow: 1000 L/Hr

Nebulizer (Bar): 7.0

Condition for MRM:

Scan type: MRM

Function type: MRM of 1 channel

Table 3: MRM of 1 channel.

Preparation of diluent for L-methyl glyoxalate monohydrate

Prepare a degassed mixture of Methanol, Water and methanolic sodium hydroxide solution in the ratio of 800:200:0.1 v/v/v.

Preparation of diluent for 2-methoxy 4-amino-5-fluro pyramidine

Prepare a degassed mixture of Acetonitrile, Water and Formic acid in the ratio of 500:500:0.1 v/v/v.

Preparation of standard solution for L-methyl glyoxalate monohydrate

Primary standard stock solution

Accurately weigh and transfer about 11.0 mg of L-Menthyl Glyoxalate reference sample into a 50 mL clean, dry volumetric flask, add 30 mL of diluent sonicate to dissolve and make up to volume with diluents (0.22 mg/mL).

Pipette 3.0 mL of Primary standard stock solution into 50 mL of volumetric flask make up to the mark with diluents (528.0 µg/g). Further pipette 3.0 mL of solution into 50 mL of volumetric flask make up to the mark with diluents (31.68 µg/g). Further pipette 2.0 mL of solution into 10 mL of volumetric flask make up to the mark with diluent (6.34 µg/g).

Preparation of standard solution for 2-methoxy 4-amino-5-fluro pyramidine

Primary standard stock solution

Accurately weigh and transfer about 9.8 mg of 2-Methoxy 4-Amino-5-Fluro pyramidine reference sample into a 50 mL clean, dry volumetric flask, add 30 mL of diluent sonicate to dissolve and make up to volume with diluents (0.196 mg/mL).

Pipette 2.0 mL of primary standard stock solution into 50 mL of volumetric flask make up to the mark with diluents (784.0 µg/g). Further pipette 2.0 mL of solution into 50 mL of volumetric flask make up to the mark with diluents (31.36 µg/g). Further pipette 2.0 mL of solution into 10 mL of volumetric flask make up to the mark with diluent (6.27 µg/g).

Preparation of sample solution for L-methyl glyoxalate monohydrate

Accurately weigh and transfer 250 mg of sample into a clean, dry 10 mL volumetric flask/Centrifuge tube, add 5.0 mL of diluent sonicate to dissolve and then add 5.0 mL of diluent and mix well. Total volume is 10.0 mL [8-10].

Preparation of sample solution for 2-methoxy 4-amino-5-fluro pyramidine

Accurately weigh and transfer 100 mg of sample into a clean, dry 10 mL volumetric flask/Centrifuge tube, add 5.0 mL of diluent sonicate to dissolve and then add 5.0 mL of diluent and mix well. Total volume is 10.0 mL [11-13].

Results and Discussion

Validation

System precision: Prepared the standard solution of L-menthyl glyoxalate and 2-methoxy 4-amino 5-fluoropyrimidine as per methodology.

Table 4: L-menthyl glyoxalate and 2-methoxy 4-amino 5-fluoropyrimidine.

%RSD for six standard solution injections were observed below 10.0% for both impurities and results are 1.8% for L-menthyl glyoxalate and 3.2% for 2-methoxy 4-amino 5-fluoropyrimidine. System precision met the acceptance criteria.

Identification and specificity

The identification and specificity is defined as the ability to assess and ensure that the impurities and diluent do not affect the sample analyzed.

For L-methyl glyoxalate monohydrate

Inject the blank (as diluent), standard solution, and control sample, spiked sample with all related compounds with and without LMGM and at specification level. Check the interference at the retention time and mass of analyte.

For 2-methoxy 4-amino-5-fluro pyramidine

Inject the blank (as diluent), standard solution, and control sample, spiked sample with all related compounds with and without MAFP and at specification level. Check the interference at the retention time and mass of analyte.

Results table for identification

Table 5: Table for Identification.

Results table for Specificity

Table 6: Table for Specificity.

Spiked sample Retention Times (RT) were comparable to that of reference sample for both impurities and both methods. Did not show any response at the retention time of the L-methyl glyoxalate monohydrate and 2-methoxy 4-amino-5-fluro pyramidine respective methodologies. Hence methods can be capable for the identification and proved the specificity.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Prediction of LOD and LOQ

Prepared a series of diluted solutions of L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities with respect to analyte concentration were prepared and injected into the LC-MS/MS system until to get the signal to noise (USP S/N) ratio is more than 3 for LOD and more than 10 for LOQ and achieved the target. The Signal to Noise (S/N) ratio of L-menthyl glyoxalate and 2-methoxy 4-Amino-5-Fluro pyramidine peaks were recorded using system data software. The LOQ and LOD results were captured in the below tables. Results Limit of Quantitation and Limit of detection.

Table 7: Limit of quantitation and limit of detection.

Precision at LOD and LOQ and accuracy at LOQ

Six different solutions were prepared to contain L-menthyl glyoxalate impurity and 2-methoxy 4-amino-5-fluro pyramidine impurity separately as per methodology at proposed LOD and LOQ level and each solution was injected once into the L-MS/MS, area of L-menthyl glyoxalate impurity and 2-methoxy 4-amino-5-fluro pyramidine impurity in each solution was recorded. %RSD for the content of L-menthyl glyoxalate impurity and 2-methoxy 4-amino-5-fluro pyramidine impurity in each preparation was calculated. The % recovery of each solution was calculated.

Table 8: Area of L-menthyl glyoxalate.

Table 9: Area of Spiked Sample (L-Menthyl Glyoxalate).

Table 10: Area of Spiked Sample (2-Methoxy 4-Amino-5-Fluro pyramidine).

S/N ratio, %RSD and Accuracy results were met the acceptance criteria.

Linearity

Seven linearity solutions were prepared for L-Menthyl Glyoxalate and 2-Methoxy 4-Amino-5-Fluro pyramidine impurities from LOQ to 150% with respect to test concentration (Table 11 and Figure 5, Table 12 and Figure 6).

Table 11: L-menthyl glyoxalate

Figure 5: Linearity Plot for L-menthyl glyoxalate.

Table 12: 2-methoxy 4-amino-5-fluro pyramidine.

Figure 6: Linearity Plot for 2-Methoxy 4-Amino-5-Fluro pyramidine.

The correlation coefficient is more than 0.99 for L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities in both methodologies. Hence the response of for L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities is linear LOQ to 150% of specification level in both methods.

Method precision

Six sample solutions were prepared individually by spiking the impurity into the Emtricitabine as per test method at specification level and injected into LC-MS/MS as per methodologies (Tables 13 and 14).

Table 13: Sample Preparation.

Table 14: Preparation Method precision.

%RSD of area and obtained concentration for method precision met the acceptance criteria as per set criteria is not more than 20.0% for both L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities.

Accuracy

Prepared the samples spiked with L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities at 100% level and 150% level in presence of Emtricitabine drug substance (prepared in triplicates) against L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluoropyrimidine impurities at 100% level and 150% level in absence of Emtricitabine drug substance and injected into LC-MS/MS as per methodologies (Tables 15-18).

Table 15: 100% of spiked sample (L-menthyl glyoxalate).

Table 16: 150% of spiked sample (L-menthyl glyoxalate).

Table 17: 100% of spiked sample (2-methoxy 4-amino-5-fluro pyramidine).

Table 18: 150% of Spiked Sample (2-Methoxy 4-Amino-5-Fluro pyramidine).

The individual percentage recovery for each sample at 100% level & 150% specification level are meeting set criteria of between 80.0% to 120.0%.

Range

Range of analytical method can be obtained from linearity and recovery data of L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities in both methodologies. Reported the range in LOQ to 150% with respect to specification level.

Robustness

Prepared standard solution as per test methods for L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine impurities at specification level. Injected to in to LC-MS/MS at deliberately varied conditions to evaluate the system suitability and methods ability to remain unaffected (Tables 19 and 20).

Table 19: L-menthyl glyoxalate.

Table 20: 2-Methoxy 4-amino-5-fluro pyramidine.

The system suitability results at each of the varied conditions complied the requirements as per the test procedure. Hence it can be concluded that the test method is robust across the extent of changes studied for each of the above parameters.

Specimen chromatograms

The result show in Figures 7-10.

Figure 7: Blank and L-menthyl glyoxalate LCMS chromatograms.

Figure 8: Control sample and spiked sample LCMS chormatograms.

Figure 9: Blank and 2-methoxy-4-amino-5-fluoro pyrimidine LCMS chromatograms.

Figure 10: Control sample and spiked sample LCMS Chormatograms.

Conclusion

Analytical test method for determination of L-menthyl glyoxalate and 2-methoxy 4-amino-5-fluro pyramidine in emtricitabine by LC-MS/MS was validated for system suitability, identification, specificity, linearity, method precision, accuracy, range (linearity, precision and accuracy) and robustness (flow rate and source cleaning) and meets all the pre-established acceptance criteria.

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