A sensitive and rapid ultra high-performance liquid chromatography with tandem mass spectrometric assay for the simultaneous quantitation of cyclophosphamide and the 4-hydroxycyclophosphamide metabolite in human plasma

Abstract

Analysis of cyclophosphamide (CP) and its metabolite, 4-hydroxycyclophosphamide (4OHCP), in a single assay has the ability to improve sampling techniques benefitting both the patients who are receiving the drug and the clinicians drawing samples. Due to instability in plasma (t = 4 min), immediate stabilization of 4OHCP with phenylhydrazine is necessary upon sample draw. After stabilization, 4OHCP and the stable CP prodrug concentrations can be analytically measured to elucidate the pharmacokinetics, including half-life and exposure parameters (Cmax and AUC). For this purpose, a sensitive analytical method was developed to measure both the prodrug and active metabolite. A liquid-liquid extraction recovered the analytes prior to analysis with an ultra HPLC-MS/MS. A Thermo Scientific™ Hypersil™ BDS C18, 2.1 × 100 mm, 3.0 μm column was used for compound separation. Mass transitions for CP (m/z 261.0 ➔ 140.0), the internal standard d4-CP (m/z 265.0 ➔ 140.0), 4OHCP (m/z 367.3 ➔ 147.1), and the internal standard AZD7451 (m/z 383.4 ➔ 341.1) were monitored over a calibration range of 34.24-34,240 ng/mL and 3.424-3424 ng/mL for CP and 4OHCP, respectively. Each calibration range proved accurate (<15% deviation) and precise (<15% RSD) for the desired compound. Using this method, CP and 4OHCP plasma levels can be measured in clinical samples from patients receiving this therapy.

Phenotyping Study of Cyclophosphamide 4-Hydroxylation in Malay Cancer Patients

“…Blood spotting (30 µL) on DBS card must be done immediately because 4-OHCP t 1/2 in blood at 37°C is about four minutes. 30 …”

A review of bioanalytical methods for chronic lymphocytic leukemia drugs and metabolites in biological matrices

“…A simultaneous quantification of CP and 2‐dechloroethylcyclophosphamide was reported in Yang et al (); of CP and 4‐OHCP in Hall, Peer, Fitzhugh, and Figg () in human plasma; and of CP, methotrexate, ifosfamide, bendamustine, doxorubicin, daunorubicin, irinotecan, and paclitaxel in Izzo et al () from human plasma and urine. Hall et al () and in Izzo Yang et al () used UHPLC–MS/MS with multiple reaction monitoring mode for a rapid analysis, whereas Izzo et al () adopted the selected ion reaction monitoring mode to quantify the analytes. The simultaneous development and quantification of CP and fludarabine used acetonitrile‐based precipitation.…”

“…reported liquid-liquid extraction followed by a simultaneous quantification of CP, ifosfamide, irinotecan, etoposide, gemcitabine, carboplatin, and pemetrexed in human plasma; Canal-Raffin et al(2016) reported the quantification of CP, ifosfamide, and methotrexate in human urine by LC-MS/MS using multiple reaction monitoring.The LLOQ developed was 20 pg/mL in urine for CP, ifosfamide, and methotrexate. A simultaneous quantification of CP and 2-dechloroethylcyclophosphamide was reported inYang et al (2014); of CP and 4-OHCP inHall, Peer, Fitzhugh, and Figg (2018) in human plasma; and of CP, methotrexate, ifosfamide, bendamustine, doxorubicin, daunorubicin, irinotecan, and paclitaxel in Izzo et al (2018) from human plasma and urine. Hall et al (2018) and in Izzo Yang et al (2014) used UHPLC-MS/MS with multiple reaction monitoring mode for a rapid analysis, whereas Izzo et al (2018) adopted the selected ion reaction monitoring mode to quantify the analytes.…”

Development and Validation Method of Cyclophosphamide and 4-Hydroxycyclophosphamide With 4-Hydroxycyclophosphamide-D4 as Internal Standard in Dried Blood Spots Using Uplc-MS/MS

“…CP is given orally or by intravenous injection or infusion and inactive until metabolized by the liver microsomal enzymes (mainly CYP2B6 with CYP3A4, CYP2C19, and CYP2A6 also contributing) yields its active metabolite, 4-hydroxycyclophosphamide (4-OHCP). In the systemic circulation, 4-OHCP is in equilibrium with its tautomeric form, aldophosphamide, which will be hydrolyzed to phosphoramide mustard and acrolein [2][3][4][5][6][7][8][9]. The effectiveness of CP chemotherapy depends on 4-OHCP concentration in blood.…”

“…and needs to be stabilized rapidly through a derivatization reaction to prevent its hydrolisis to phosporamide mustard and acrolein [8]. Various agents have been used for this reaction, including semicarbazide [4][5][6], aryl hydrazines [7][8][9], sodium cyanide hydrazone [10], and hydroxylamines [11,12]. In this study, we used semicarbazide as the derivatization agent.…”