HPLC-MS/MS method development and validation for the determination of tetradecapeptide in human plasma

Abstract

Introduction. The number of peptide drugs being developed and registered has increased in recent years. Therefore, modern analytical approaches and methods are required to determine these substances in biological matrices during pharmacokinetic studies. Peptides are structurally intermediate between small molecules and biopolymers, making it difficult to develop methods for determining them using High Performance Liquid Chromatography with Tandem Mass Spectrometry (HPLC-MS/MS). Peptide derivatization can help achieve optimal chromatographic separation and increase method sensitivity.Aim. To develop and validate a method for the determination of the tetradecapeptide (TDP) threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate in human plasma by HPLC-MS/MS.Materials and methods. The determination of TDP in human plasma was performed by HPLC-MS/MS. Sample preparation included a combination of blood plasma protein precipitation with propionic acid solution in methanol, liquid-liquid extraction with chloroform, and peptide derivatization with propionic anhydride. Internal standard (IS) was threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-leucyl-glutamyl-arginyl-glutamyl-lysyl-glutamate. Chromatographic separation was performed in gradient mode, eluent A was 0.1 % formic acid solution in water, eluent B was 0.1 % formic acid in acetonitrile. Column: Waters XBridge C18, 4.6 × 50 mm, 5 µm. Ionization source was electrospray in positive mode. Multiple reaction monitoring (MRM) transitions for 4-substituted TDP propionate were: 681.30 → 73.95 m/z, 681.30 → 84.00 m/z, 681.30 → 101.90 m/z, 681.30 → 140.10 m/z, and for 4-substituted IS propionate: 686.00 → 74.10 m/z, 686.00 → 84.05 m/z, 686.00 → 102.00 m/z, 686.00 → 140.00 m/z.Results and discussion. Validation of the developed method was carried out in accordance with the requirements of Eurasian Economic Union and the following parameters were determined: selectivity, matrix effect, calibration curve, accuracy and precision, recovery, lower limit of quantification, sample carryover, stability.Conclusion. The method for the determination of TDP in human blood plasma by HPLC-MS/MS was developed and validated. The analytical range was 5.00–1000.00 ng/mL, allowing the method to be used to study TDP pharmacokinetics.