https://doi.org/10.1038/s41598-022-24215-4
”
Prior to measuring hormone levels in the study samples, we used our standard protocol consistent with FDA guidelines to validate our LCMS methods for measuring LNG and MPA in serum and urine. Specifically, we spiked six concentration levels of calibration standards (STDs) in the range between 0.1 and 10 ng/mL in human serum and separately prepared three levels of quality control samples (QCs) at 0.3, 1, and 7 ng/mL concentrations. We used LNG-d6 or MPA-d6 (5 ng/ml) as the internal standards (IS) respectively and evaluated and validated the following parameters: specificity, linearity, limit of quantitation, accuracy, precision, matrix effects, recovery, and stability. Our results showed that we met the acceptance criteria of all validation parameters as per the protocol.
For the next step, we measured LNG and MPA levels in both serum and urine samples by LC-MS/MS using LNG-d6 or MPA-d6 as the internal standards respectively. To purify LNG or MPA from serum test samples we used the protein precipitation method. Specifically, we added 100 µl of methanol to 100 µl of the test sample containing the IS (5 ng/ml). We vortexed the samples for 15 mins (centrifuged at 14.5 × 103 rpm for 10 mins) and transferred the supernatants to high-performance liquid chromatography (HPLC) vials for injection. In addition, we processed the LNG or MPA standards and the quality control samples that were spiked with IS using the same protein precipitation method.
To purify LNG or MPA from urine samples, we performed solid-phase extraction using Oasis HLB Plus Light cartridges (Waters Corporation, Milford, MA). We eluted LNG or MPA from the cartridge using 1 ml of methanol and dried each sample under nitrogen gas. We reconstituted the purified LNG/MPA with 150 µl mobile phase A/B (50/50, v/v) and injected 10 µl aliquots into the autoinjector assembly. In addition, we processed LNG or MPA standards, quality control samples spiked with IS and 1 ml blank human urine using the same solid phase extraction method. We used Waters Acquity UPLC BEH C18 (1.7 µm, 2.1 × 50 mm) column to separate LNG or MPA from other components by gradient elution using the mobile phase from 80% A (2 mM ammonium formate + 0.1% formic acetic in 10% methanol) and 20 % B (100% methanol) up to 6 minutes to 90% B with 0.3 ml/min flow rate. We then used Waters TQ-s to monitor the product ion transitions of 313.35 m/z to 245.28 m/z for LNG and 319.38 to 251.32 m/z for LNG-d6 IS. Similarly, we monitored the transitions 387.42 m/z to 327.35 m/z for MPA, and 393.40 m/z to 330.34 m/z for MPA-d6 IS. We used MassLynx software version 4.2 to control all parameters of the LC–MS/MS system.
In analyzing study samples, we included six levels of calibration standards, one double blank, one matrix blank spiked with internal standard, and three levels of quality control samples (n = 2). We placed one set of quality control samples in the front of the sample queue and another set at the end of the sample queue. The calibration standards were injected at the front and reinjected at the end sample queue. The lower limit of quantitation (LLOQ) for serum LNG and MPA was 0.1 ng/ml and for urine LNG and MPA was 25 pg/ml. A 50 µl of the serum sample was used for LNG or MPA in serum assay and a 1 ml human urine sample was used for LNG or MPA in urine assay. Intra- and Inter-assay coefficient of variations was less than 15% based on the results of quality control samples.
“