An LC–MS/MS method development and validation for the quantification of selpercatinib in spiked human K2EDTA plasma

2.1. Chemical and reagents

Obtaining selpercatinib from Jinan Million Pharmaceutical Co., Ltd. in India proved to be successful. During the course of the investigation, an HPLC-water purification system manufactured by Millipore and located in Bedford, United States of America was employed. To get the internal standard for ibrutinib (IBTB), MSN laboratories in India were acquired. The reagent grades of formic acid, methylalcohol, and acetonitrile were acquired from A.B enterprise in Mumbai, India. The methanol was of the HPLC quality. Obtaining drug-free plasma that included K2EDTA anti-coagulant was accomplished by Sai Laxmi Clinical, which is located in Hyderabad, India.

2.2. Instrument

The current investigation made use of the Shimadzu LC20AD prominence liquid chromatography system with SIL/HTCautosampler, DGU20A3 prominence degassing system, and Applied Biosystems MDS/SCIEX API 6500 mass spectrometric system, located in Japan. Chromatograms from the chromatographic system were acquired using the 1.4.2 version of the Analyst program.

2.3. Processing of quality control and calibration standards

The stock solutions of selpercatinib and IS were executed by solubilizing the reference standards in diluent methyl alcohol and ACN (1:1 %v/v) to produce concentrations of 0.2 g/ml. Resulting solutions were carefully monitored at temperatures below −20°C in a freezer. The analyte and internal standard (IS) were diluted in the diluent to create working solutions. The solutions were put away at the temperature of the room and processed on a daily basis. The calibration standards underwent processing by adding selpercatinib to blank plasma, resulting in concentrations of 93, 182, 315, 615, 1,085, 1,625, 2,165, and 2,710 ng/ml. The samples for quality control were processed at various concentrations to ensure accuracy and precision in the analysis of selpercatinib. These concentrations included an LLOQ QC, LQC, MQC, and HQC. Separate portions of the bulk spiked samples were placed in polypropylene tubes and kept at a temperature of −70°C. After thawing to room temperature, all frozen calibration standards and QC samples were processed in preparation for the analysis.

2.4. Chromatographic parameters

Processed samples were separated chromatographically using a Zorbax SB-C18 column (50 mm × 4.6 mm) 3.5 µm with a mobile phase of acetonitrile, methyl alcohol, and 0.1% HCOOH in the proportion of 20:70:10. The moveable solvent system was measured using a column with 0.7 ml/minute rate of flow. The autosampler was retained at 5°C with a 10 μl infusion volume. At a temperature of 35°C, the analytical column was seen in the oven. The drug and IS were separated in 6.0 minutes total.

2.5. Conditions for mass detection

While the transitions of m/z findings of 526.25/450.16 for selpercatinib and 441.2/55.01 for ibrutinib were analyzed using an MRM mode, mass quantitation was performed on the transitions. Mass instrument was run in the positive ionizing method, with the DP (declustering potentials) values being 30V and 50V, and the CE (collision energies) findings being 35 and 18V for selpercatinib and ibrutinib, respectively. Pressures of the nebulizer gas (GS1), collision gas, curtain gas, and auxiliary gas were retained at 30 psi, 20 psi, 50 psi, and 55 psi, correspondingly during the duration of the experiment. The temperature of the ion spray was maintained at 450°C, while the voltage of the ion spray was maintained at 4,500V.

2.6. Processing of sample solution

A small portion of the 300 μl plasma sample was relocated to the 5 ml polypropylene tube. After that, 10 μl of IS (100 ng/ml) solution was added and the tube was vortexed. Then, 500 µl of buffer was added to the solution, and it was subjected to centrifugation at 15,000 rpm for 25 minutes at 5°C. The organic layer was then dried with an evaporating unit, and 300 µl of the mobile solvent system was added to the dry material that was left over. The solution was then put into autosampler tubes so that it could be infused into the chromatographic machine.

2.7. Analytical method validation

The developed technique for measuring selpercatinib was accepted by the USFDA following their guidelines for bioanalytical method evaluation [10–12]. It was necessary to show that the parameters were stable, accurate, linear, selective, residual, precise, recovered, and had a matrix effect.

3.1. Method optimization

3.1.1. Mass spectrum conditions

We used the positive ionizing method in MRM with selpercatinib to figure out the mass, which gave us more sensitivity and good precision. The standard solutions were pumped into a mass system through the syringe pump to find precursor and product ionic components. The mass spectrum of the IBTB and selpercatinib product ions was found to be 55.01 and 450.16, which were chosen as a measurement of ionic components. At the same time, the mass spectrum settings’ factors were fine-tuned to get a better mass reaction. These included the voltage of capillary, voltage of ionic spray, temperature of ionic spray, and gases related to heating, nebulizing, colliding, curtain, and more [13–15].

3.1.2. Chromatography

The column temperature, organic phase, and movable phase (varying concentrations of ammonium acetate and of HCOOH) were tuned for liquid chromatography to provide superior peak shape, increased sensitivity, and no matrix impact. Furthermore, the methyl alcohol-ACN organic phase was chosen as a mobile solvent system over water because of its strong elution impact and minimal background noise. Various trials were executed with HCOOH, methyl alcohol, ACN, and 10mM ammonium acetate, and finally with a Zorbax SB-C18 column (50 mm × 4.6 mm) 3.5 µm with a mobile phase of acetonitrile, methyl alcohol, and 0.1% HCOOH in the proportion of 20:70:10. The moveable solvent system was measured using a column with 0.7 ml/minute rate of flow and at a column oven temperature of 35°C.

3.1.3. Selection of IS

In a current investigation, IBTB was chosen as an IS because it exhibited comparable chromatographic properties, extraction efficiency, ionization, and retention times to selpercatinib. According to the technique validation findings, no evident interferences were identified at analyte and IS retention periods [16–18].

3.2. Validation of the method

3.2.1. Specificity

Plasma samples from six different batches of human plasma were spiked with selpercatinib at the LLOQ and IS to estimate specificity. According to Figure 2, the retention times of selpercatinib and IBTB were approximately 2.6 and 4.3 minutes, correspondingly [19]. There were no apparent interferences from any natural substances or ISs on the analytical results of selpercatinib. Additionally, the responses of all noisy peaks were found to be <20 % of samples with the LLOQ. Meanwhile, the LLOQ was measured with accuracy that met acceptable standards, demonstrating precision below 20% and a signal-to-noise ratio (S/N) above 5. In the analytical run, the highest concentration of calibration standard was immediately infused into LC-MSMS after blank human plasma for carry-over effects assessment. Upon further observation, no discernible carry-over effect was detected.

Figure 2. (A) Blank plasma and (B) spiked LLOQ-sample chromatograms of selpercatinib. [Click here to view]

3.2.2. Sensitivity and calibration curve

The established method for determining selpercatinib exhibited excellent linearity, covering a range of 93–2710 ng/ml. The linearity curves were made using peak response ratios of selpercatinib to IBTB against the concentration with 1/C² weighting factors [20]. The calibration curves’ regression line equation is y = 0.0012x - 0.0378, indicating a strong correlation coefficient >0.99 (Fig. 4 and Table 1). LLOQ of selpercatinib was determined to be 93 ng/ml, with a S/N ratio >5. This analysis was conducted using five replicates, ensuring the reliable quantitation of selpercatinib in plasma samples studied.

Figure 3. (A) LQC, (B) MQC, and (C) HQC sample chromatograms of selpercatinib. [Click here to view]

Figure 4. Linearity graph of selpercatinib. [Click here to view]

Table 1. Selpercatinib data for calibration controls.

RE- Relative error; LS- Linearity standard; RSD- Relative standard deviations.

3.3. Precision and accuracy

The precision and accuracies of intra and inter-batches were assessed by analyzing six spiked plasma samples of selpercatinib at the lower limit of quantification and three quality control levels in a single batch and in three successive batches, correspondingly. The precision and accuracy findings for the quantification of selpercatinib can be found in Table 2. The precision deviation values for intra and inter-batches were varied between 2.41 and 5.23 of relative standard deviations. The accuracy deviation findings for intra and interbatches varied from −4.43 to 5.3 of relative error [21–24]. The findings clearly indicate that the precision and accuracy of determining selpercatinib in plasma were consistently reliable and reproducible.

Table 2. Inter and Intra-batch precision and accuracy.

3.3.1. Extraction recoveries

Proper pretreatment was performed on the biosamples prior to detection. A peak area ratio between the extracted spiked samples and 3 QC standard samples(n = 6) at matching concentrations was used to assess the selpercatinib extraction recoveries. In a similar vein, the peak response ratio of spiked samples of plasma at matching concentration levels to QC plasma sample solutions(n = 6) was used to assess the extraction recovery of IS [22]. At MQC, LQC, and HQC levels (Fig. 3), the average selpercatinib extraction recovery was 90.8%, 93.43%, and 88.74%, respectively. The average recovery rate of IS extraction at a concentration of 100 ng/l was 95.67%. In Table 3, you can see the outcomes.

Table 3. Selpercatinib and IS extraction recoveries data.

RSD- Relative standard deviation; X, average recoveries of unextracted solutions; Y, mean recoveries of extracted solutions.

3.3.2. Dilution integrity

The dilution integrity test was done with all three analytes at 2 times the upper limit of quantification concentration. We found that the percentage of the dilution QC sample solutions was in a range of 85.0% to 115.0 % of the original figure after a 1:4 dilution, with a %RSD of ≤5.2 [18,25,19]. In the same way, LQC samples that were spiked with a drug at the same time were measured within 15% of the standard value, with a %RSD of 4.6.

3.3.3. Matrix effects

Because the test is so accurate, co-eluting matrix constituents can either stop or boost ionization, but blank matrixes might not show any response [16–18,25]. Therefore, the possibility of varying matrix relating ion destruction was tested in eight sources of human plasma, two of which were hemolytic and two of which were lipemic. This was done by finding the IS normalized matrix factor. Table 4 shows that an average IBTB adjusted matrix factor for all the analytes was between 0.943 and 1.11, with a standard deviation of 4.98%.

Table 4. Selpercatinib matrix effects data.

Relative standard deviation; B, Batch; a, Hemolyzed lot; b, Lipemic lot; MF, matrix factor; RSD.

3.4. Stability studies

For the purpose of determining stability, both matrix-based and aqueous sample solutions were subjected to tests. Analyte and IS in the stock solution were stable for 66 days between 1°C and 10°C, while stock dilutions in diluent were stable for up to 48 hours at any temperature between 1°C and 10°C [8,9,20]. Both −70°C and −20°C were used to achieve stability in the matrix for a period of 63 days. Table 5 presents the findings of the stability assessments that were conducted.

Table 5. Selpercatinib stability data.

Y, average drug concentration; X, nominal concentration of drug.

An examination of the matrix’s stability was carried out in comparison to recently spiked calibration standards. At a temperature of less than 10°C and after six cycles of freezing and thawing, the drug remained stable for 16 hours on the bench. Up to 47 hours in the autosampler at 10°C, the processed samples remained stable. Over the course of the stability length and circumstances, there was no identification of any substantial degradation or inter-conversion of the analyte. The average response ratio of stability solutions was compared to the response ratio of reference samples to assess the stability of whole human blood at both low and high-quality control levels. Above 10°C, the analyte remained stable in whole human blood for up to 2.5 hours.