Solvents and chemicals

The amivantamab sample (99.99% purity) was provided as a gift sample from Shree Icon Labs, Vijayawada, Andhra Pradesh, India. All other chemicals, including HPLC grade acetonitrile and methanol, were purchased from Merck Chemical Division in Mumbai. The Milli-Q water purification system's HPLC-grade water was used throughout the study. All chemicals and reagents were used as received without further purification. Trastuzumab (99.98% purity) was used as an internal standard (IS). It was obtained from Glenmark Pharmaceuticals Pvt Ltd, Mumbai, India.

Instrumentation

The liquid chromatography system comprised of Waters, alliance e-2695 model HPLC armed with column oven, autosampler, and degasser was employed for analysis. The SCIEX QTRAP 5500 mass spectrometer was connected to the HPLC system. REMI centrifuge was used for centrifugation. Cyclo Mixer was used for mixing.

Chromatographic conditions

The autosampler was maintained at ambient temperature. An Agilent eclipse C18, 150 mm × 4.6 mm, 3.5 µm column was used for elution. Isocratic elution was employed with a mobile phase comprising acetonitrile and 0.1 M ammonium formate in the proportion of 40:60. With an injection volume of 10 µl, the chromatographic flow rate was set at 1.0 ml/min.

Mass spectrometer conditions

The mass spectrometric system comprised a SCIEX QTRAP 5500 mass spectrometer armed with an electrospray ionization interface ionization mode. The collision gas used was nitrogen. The compound-specific parameters working are displayed in Table 1. The ions were detected using multiple-reaction monitoring mode (MRM). Sciex Analyst software was used to process the data.

Table 1 Compound-specific parametersSelection of internal standard (IS)

Trastuzumab was selected as an internal standard to reduce the inaccuracy at the processing level or the ongoing analysis level caused by the instrument for quantifying both analytes by technique. Trastuzumab was chosen as the IS due to its near molecular weight with the analyte.

Preparation of solutionsPreparation of amivantamab stock and working solutions

Five milligrams of amivantamab working standard was weighed and transferred into a 100-ml volumetric flask and then diluted to volume with diluent. Further, 1 ml from the above solution was transferred to a 10-ml volumetric flask and made up with diluent. 0.4 ml of the above solution was taken into a 10-ml volumetric flask and made up to the mark with diluent.

Preparation of internal standard stock solution (200 ng/ml)

Five milligrams of trastuzumab working standard was weighed and transferred into a 100-ml volumetric flask and, then, diluted to volume with diluent. Further, 1 ml was pipetted into a 10-ml volumetric flask and made up with diluent. 0.4 ml of the above solution was taken into a 10-ml volumetric flask and made up to the mark with diluent. This IS added to the sample processing (50 µl) resulted in a final concentration of 50 ng/ml corresponding with the MQC concentration of amivantamab.

Preparation of plasma samples

Transferred 500 µl of amivantamab stock solution into a 2-ml Eppendorf tube. To this, 200 µl of plasma, 500 µl of internal standard, 300 µl of acetonitrile, and 500 µl of diluent were added (50 ng/ml). Calibration standards were prepared by spiking blank rat plasma with appropriate amounts of amivantamab and trastuzumab. Calibration standards for final concentration were 5.00, 12.50, 25.00, 37.50, 50.00, 62.50, 75.00, 100.00 ng/ml for amivantamab. The preparation of calibration standards and QC samples are displayed in Tables 2 and 3.

Table 2 Preparation of standardsTable 3 Preparation of QC samplesDiluent

Mobile phase of acetonitrile and ammonium formate in the ratio 40:60 was used as diluent.

Sample preparation

The 2000 µl samples prepared above were vortexed with the vortex cyclo mixture. The solution was centrifuged at 4000 RPM for 15 to 20 min. Then, the supernatant fluid was collected in an HPLC vial. Double blank samples (i.e., without analyte and IS) were prepared by mixing 1000 µl of acetonitrile with 200 µl of rat plasma samples.

Bioanalytical method validation

Method validation was done according to ICH M10 guidelines.

System suitability

Six replicates of high-quality control (QC) standard solution were injected into the chromatographic apparatus to analyze the system suitability parameters.

Linearity

The calibration curve was generated by analyzing eight concentrations of amivantamab in plasma. Samples were measured by comparing the peak area of amivantamab to that of trastuzumab. The plot of peak area ratios vs plasma concentrations was made.

Specificity

The specificity of the developed approach was evaluated in the current study by analyzing the chromatograms of blank plasma and spiked plasma samples (amivantamab, trastuzumab).

Sensitivity

The method’s sensitivity was assessed by analyzing 6 replicates of rat plasma comprising a lower limit of quantification (LLOQ) sample (5.0 ng/ml) of amivantamab.

Accuracy and precision

Four QC samples—LLOQ, low-quality control (LQC), medium-quality control (MQC), and high-quality control (HQC)—each with six replicates were used to assess the proposed bioanalytical method's precision and accuracy. The precision and accuracy of the proposed method were represented as mean accuracy (%) and coefficient of variance (CV) (%), respectively.

LOD and LOQ

The signal-to-noise ratio was used to calculate the bioanalytical method's limit of detection (LOD) and limit of quantification (LOQ).

Autosampler carryover

Autosampler carryover was assessed by injecting a blank sample, followed by an HQC sample and then an LLOQ sample followed by a blank sample.

Dilution integrity

The dilution integrity of plasma samples was evaluated by evaluating amivantamab samples above ULQC. Those samples were taken and diluted with a blank matrix to MQC and ULQC.

Matrix effect

The matrix effect of plasma on the response of amivantamab was assessed by blank plasma samples that were extracted from six different lots and reconstituted to form working standards of LQC and HQC. These samples in triplicate were quantified against the calibration curve.

Recovery of analyte

Six replicates of the amivantamab QC low-, medium-, and high samples were created by spiking the relevant concentrations of the drug and an internal standard into either unextracted or supernatant-recovered blank rat plasma (extracted). Recovery was calculated by comparing its response in multiple samples to neat standard solution responses.

Recovery of internal standard

Trastuzumab (50.0 ng/ml) samples in blank plasma were prepared and examined in six repetitions.

Ruggedness

Low, medium, and high QCs of amivantamab in plasma samples were reinjected into the system. % CV and accuracy were assessed to determine ruggedness.

Reinjection reproducibility

Low, medium, and high QCs of amivantamab in plasma samples were reinjected into the system. % CV and accuracy were assessed to determine reinjection reproducibility.

Stability studiesBenchtop stability

Amivantamab's stability in rat plasma was assessed by exposing six replicates of three different concentrations (LQC, MQC, and HQC) for 8 h on a benchtop and injecting them into the system.

Short-term and long-term stability

Short-term and long-term stability was assessed for amivantamab. Three different analyte concentrations were spiked into six duplicates of rat plasma for QC. LQC, MQC, and HQC samples were prepared and stored at 5 ± 3 °C for 7 days, and short-term stability was assessed. LQC, MQC, and HQC samples were prepared and stored at − 20 ± 3 °C. These samples were injected from day 1 to 28 days for every seven days (as day 1,7,14 21, and 28), and long-term stability was assessed.

Freeze–thaw stability

The stability of amivantamab was evaluated after freeze–thaw cycles, respectively. Each LQC, MQC, and HQC had six duplicates that were held at − 20° C, totally thawed at ambient temperature, and then immediately refrozen at − 20° C. After this cycle was done twice, the samples were removed for injection into the LC-MS.

Autosampler stability

LQC, MQC, and HQC samples of amivantamab in plasma were injected at one-hour up to 24-h intervals. Mean accuracy (%) and CV (%) were calculated.

Dry extract and wet extract stability

Wet extract stability was evaluated by assessing the six sets of LQC, MQC, and HQC after 12 h and 18 h that were stored at 2–8 °C. The dry extract stability test used six sets of LQC, MQC, and HQC after 12 h and 18 h that were stored at 22 °C.

Assay

Assay was done to define the applicability of the bioanalytical method to the marketed formulation. MQC (50.0 ng/ml) sample was prepared from the marketed formulation (Rybrevant) and injected into the LC-MS system.

Method applicability to rats

Six healthy white female albino rats (body weight in between 250 and 350 g) were taken from Flair Labs, Gujarat, India. Before the experiment was directed, rats were adapted to laboratory environments for seven days. Diet was restricted for 12 h before the experiment, although water was freely available. A single dose of amivantamab (0.83 mg/ml) was administered to six rats. Samples were collected at different intervals, such as 10, 20, 30, 40, 50, and 60 min. K2 EDTA vacutainer tubes were used to collect blood at each interval. A predose sample was also taken to check for any potential plasma interferences. The plasma was obtained by centrifuging the collected samples kept at 10 °C. The liquid–liquid extraction method was used to isolate amivantamab in rat plasma. The animal study protocol was approved by the Institute of the Animal Ethics Committee (Reg.No: 1250/PO/RcBi/S/09/CPCSEA). Phoenix Win Nonlin (Version 5.2) software was used to analyze the data.