Analytical Method Development and Quantification of Genotoxic and Carcinogenic Impurities in Abacavir and Lamivudine Tablets Using Hyphenated Technique

Lamivudine and abacavir are two quite well-known antiretroviral drugs prescribed in HIV/AIDS. Sometimes these two medications are used together because they significantly reduce virus proliferation and enhance patient results. Like any medication, Abacavir and Lamivudine pills must be flawless, especially in terms of those that could endanger human health. Among these poisons, genotoxic and carcinogens particularly concern me because, even at extremely low levels, they might harm DNA or potentially induce cancer. [3-8]. Identification and control of such contaminants guarantees quality, safety, and efficiency of the final medical product. While genotoxic toxins (GTIs) may change genetic material and cause mutations or chromosomal damage, carcinogens (CIs) are substances that help cancer to develop. Contaminants may arise from raw materials or the manufacturing process during the synthesis of active pharmaceutical ingredients (APIs). [9-12] Due to their potential danger, organizations like the U.S. FDA and the International Conference on Harmonization (ICH) [1-2] have established strict rules for how much of certain chemicals can be in pharmaceutical drugs. Mostly depending on a sensitive and exact analytical method for patient safety, determining and spotting genotoxic and carcinogenic elements in Abacavir and Lamivudine tablets depends. Usually used as standard analytical techniques, hyphenated methods including Gas Chromatography-Mass Spectrometry (GC-MS) enough to identify these low-level contaminants. [13-16] These very sensitive, specific, and precise techniques let one find even microscopic amounts of pollutants. This study intends to employ hyphenated techniques to create and verify an analytical approach for the identification and quantification of genotoxic and carcinogenic components in Abacavir and Lamivudine tablets. This initiative aims to guarantee the general safety and quality management of numerous significant antiretroviral drugs by means of free from dangerous chemicals possibly compromising patient health.

MATERIALS AND METHOD:

1. MATERIALS:

1.1 Chemicals:

NDMA, NDEA, and methanol, which come from Merck Life Science, are important when talking about possible flaws in drugs like Abacavir and Lamivudine pills.

1.2 Instruments:

• Gas Chromatograph equipped with programmable temperature, flow controller and MS detector (Shimadzu GC-2010 with MS detector)
• Liquid Auto sampler (Shimadzu AOC-20i Auto sampler)
• Data handling system (GCMS Solution version 2.61)
• Fused silica capillary column Rxi-1ms; 60 m long; 0.25mm internal diameter, coated with 100% Di methyl polysiloxane stationary phase of 0.25 µm film thickness.

2. METHOD [17-23]:

1. Method development:

Major important parameters in method development for GCMS was diluent (solvent), column, Oven programming, other gas chromatographic parameters like flow, injector temperature, detector temperature and mass spectrometer parameters. As the analyte of interest NDMA and NDEA were volatile in nature. The GC-MS liquid injection technique was decided for analysis due to low limit. Sample matrix was 40mg/1 mL. Due to high sample concentration the column might be damaged. It is also very important to protect the column from high concentration. It was resolved by using solvent in which API was insoluble and specifically analyte of interest were soluble. Because of this approach interference, due to API matrix can be distant. Diluents used for developmental trials were Dichloromethane, Methanol, Isopropanol, n- Hexane. Based on factors like recovery and interference of matrix at the elution time of analyte, n-Hexane/Methanol was diluent used in standard as well as sample preparation. Columns used for the developmental trials were DB-5, DB-1, DB-624 and Rtx-1301 with different dimensional parameters. Based on chromatographic response, specificity and lesser baseline interference, Rxi-Ims column, 60 m length, 0.25 mm ID and film thickness 0.25 µm is found fit for the requirement.

2. GC conditions:

The separation of the various compounds was accomplished using an Agilent J&W DB-EUPAH column (20 m × 180 μm × 0.14 μm). The GC parameters for analyzing samples were optimized based on published analytical procedures. A volume of 1 μl of the sample was injected in pulsed split less mode at 40 psi until 0.5 min. The inlet temperature was maintained at 300 degree Celsius and UHP helium was used as the carrier gas at a flow rate of 1.0 mL/min. The GC oven temperature started at 500C (0.8 min hold) and was ramped to 1800C at 700C/min (0 min hold), then to 2300C at 70C/min (1 min hold), 2800C at 400C/min (1 min hold), and finally 3300C at 250C/min (5 min hold). This resulted in a total run time of 20.05 min. The post run back flush was performed at 3300C for 4 min.

3. MS Condition:

Changing the mass spectrometry (MS) parameters for the measurement of Abacavir and Lamivudine tablets resulted in one got excellent sensitivity and accuracy. While the electron ionizing (EI) source temperature was set at 320°C, quadrupoles 1 and 3 maintained at 150°C. Nitrogen was the collision gas; helium the quench gas at 1.5 mL/min and 2.3 mL/min comparable flow rates. The SCAN method defined molecules across a mass range of m/z 50-500 using a gain factor of 10 to improve signals. This technique enables one to identify target molecule retention times and detect precursor ions. Measurement was made possible by the selected ions in the chosen ion monitoring (SIM) method matching the molecular weights of the target pollutants. The chosen stay times provide consistent peak observations ranging 30 to 50 ms. The method further employed the multiple reaction monitoring (MRM) and PMRM approaches to increase sensitivity with the precursor ions and collision energy levels optimal for the best signal-to Noise ratios. Using the APCI source to increase sensitivity and choosing the suitable retention duration for API peaks helps to avoid system contamination during pharma product analysis.

• Diluent: n-Hexane as diluent.
• Blank: n-Hexane as a blank.
• Standard Solution: Accurately weighed and transferred 0.1 g NDMA, NDEA into 100 mL volumetric flask containing 50 mL of diluent, then made up to the volume with diluent. Further, 0.160 ml of above solution was diluted in 100 mL flask with diluent. Further, 1.0 mL of above resulting solution was made up to 100 mL with diluent. Then further 5.mL of above solution was made up to 50 mL with diluent (Concentration 0.0016 µg/g).
• Sample preparation: Accurately weighed and transferred about 0.200 g of the sample, add 5.0 mL of diluent to it and shaken for 5 minutes, then kept it for standby. The undissolved sample matrix will settle down and then supernatant liquid taken in a GC vial for injection. The sample concentration is 40mg/1mL.

5. Method validation [27-29]: Many crucial criteria ensure the reliability and accuracy of the approach by means of validation for GC-MS in Multiple Reaction Monitoring (MRM) mode.

Specificity: The specificity of the approach is examined to confirm its ability to separate target analytes from probable interferences. This is reached by verifying that no overlapping peaks result from the analyte retention times.

Linearity: Good correlation coefficient (R² > 0.99) is confirmed to test linearity by means of standard solutions of target pollutants at different concentrations; response is plotted against concentration.

Limit of Detection (Lod) and Limit of Quantification (LOQ): Using the signal-to noise ratio enables one to determine the limit of detection (LD) and limit of quantitation (LOQ), hence calculating the lowest concentration at which the analyte can be regularly detected and quantified.

Precision: Finding results expressed as Relative Standard Deviation (RSD), where values around 2% are often acceptable, accuracy is evaluated by injecting the same standard solution numerous times (system precision) and evaluating different sample preparations (method precision).

Recovery: Recovery tests evaluate accuracy with usually acceptable results falling between 90 and 110% by introducing known analyte concentrations into matrix samples and computing the recovery percentage.

Sensitivity: Finding the signal to Noise ratio (S/N) at concentrations around the LOD and LOQ will enable one to assess sensitivity; higher S/N ratios suggest better sensitivity. Determining possible deterioration also guarantees the analyte's and standard solution's stability throughout time.

Robustness: Robustness supports the reliability of the approach under very various experimental conditions, including temperature or flow rate variations. Selectivity shows that from interfering molecules in the sample matrix; the method does not generate noticeable reactions. System suitability checks ensure that the instrument performs as anticipated before every batch analysis, thereby evaluating parameters like resolution, peak symmetry, and retention time precision. These validation procedures taken together ensure that the GC-MS method in MRM mode is accurate, consistent, and exact for genotoxic and carcinogenic contamination of pharmaceutical products.

RESULTS AND DISCUSSION:

a) Mass spectral analysis: The peak of NDMA elutes at 3.134 minutes. NDEA elutes at 5.208 minutes. NDMA mass spectra showed fragments at m/z 74. Similarly, NDEA showed fragments at m/z 102 respectively. Spectra of both the components is compared and matched with NIST spectrum library. Due to maximum response of these m/z values used for quantification of NDMA and NDEA by Multiple reaction monitoring (MRM). Refer spectra in figure 1 and 2.

Figure 1 Spectrum of N-Nitrosodimethylamine (NDMA)

Figure 2 Spectrum of N-Nitrosodiethylamine (NDEA)

System suitability: Before every parameter, six injections of system suitability solution were injected into GC-MS to check the performance of the system as a system suitability solution.

Specificity: NDMA and NDEA individual RT check solutions were prepared and injected and confirmed the retention times and also injected other solvents used in the manufacturing process and found no interference at the elution time of impurities and is tabulated in below.

Table 2: Specificity analysis results

Detection limit (LOD) and quantification limit (LOQ) LOD-LOQ prediction: To check LOD-LOQ values, serial lowest concentration solutions were prepared, injected into the GC-MS and recorded the chromatograms. As per the ICH Guidelines considering the LOD and LOQ which is 10% and 30% of allowable intake i.e. 0.00016 µg/g and 0.00048 µg/g.

Linearity: Linearity solutions were prepared after quantitatively diluting std. stock solution to obtain solutions in the range of LOQ and 150% level of the specification level and proved that method was linear and the results are tabulated in table 3 and 4. Linearity graphs are as shown in figure 3 and 4.

Table 3: Linearity of NDMA

Figure 3. Linearity curve of NDMA

Figure 4. Linearity curve of NDEA

Table 5 System precision results

Table 6 Method precision results

Accuracy: Accuracy study was performed by spiking samples in triplicate with NDMA and NDEA at 50%, 100% and 150% level of the evaluation limits. The minimum recovery observed for NDMA was 98.20% and maximum 100.39%. The minimum recovery observed for NDEA was 97.81% and maximum recovery 100.72%. The %RSD for recovery was 0.831 for NDMA and 0.887 for NDEA and the results are tabulated in the table 7.

Figure 5 Chromatogram of Blank solution

Figure 6 Chromatogram of Standard solution at Specification concentration MRM mode (Retention time: 3.134 min: NDMA, 5.208 min: NDEA)

Figure 7 Chromatogram of LOQ level concentration

Figure 8 Chromatogram of Sample solution

Figure 9 Chromatogram of Spiked sample solution (Retention time: 3.134 min: NDMA, 5.208 min: NDEA)