Analysis of 4-Hydroxycyclophosphamide in Cancer Patients Plasma for Therapeutic Drug Monitoring of Cyclophosphamide

Abstract

Objective: To quantify 4-hydroxycyclophosphamide in cancer patients' plasma for therapeutic drug monitoring of cyclophosphamide. Methods:The blood was collected at 0.5 and 1 h after administration of chemotherapy. Prior to analysis, 4-OHCP in plasma was derivatized with semicarbazide HCl, then was extracted using 4 ml ethyl acetate and finally was determined by Ultra High-Performance Liquid Chromatographytandem mass spectrometry. Chromatographic separation was conducted using waters acquity BEH C18 column (1.7 μm; 50 mm x 2.1 mm). The mobile phase consisted of formic acid 0.1% and methanol (50: 50, v/v), column temperature 30 °C and flow rate of 0.3 ml/min. Mass detection was performed on waters xevo TQD equipped with an electrospray ionization (ESI) source at positive ion mode in the multiple reaction monitoring (MRM). Cyclophosphamide was detected at m/z 260.968>139.978, 4-hydroxycyclophosphamide-semicarbazide at m/z 338.011>224.97, and hexamethylphosphoramide as internal standard at m/z 180.17>92.08. Results:The method was linear in the range of 5-1000 ng/ml for cyclophosphamide and also for 4-hydroxycyclophosphamide. The results showed that the level of 4-OHCP in 39 cancer patients was in the range of 5.02 ng/ml to 832.44 ng/ml.Conclusion: 4-hydroxycyclophosphamide was detected on 39 patient samples with the lowest level of 5.40ng/ml and the highest level was 832.44 ng/ml. This can be a parameter that the regiment of cyclophosphamide was effective.

Method Development on Analysis Cyclophosphamide and 4-Hydroxycyclophophamide in Dried Blood Spot and Its Application in Breast Cancer Patients

“…Acquity BEH precolom was used for analysis. The mobile phase used was 0.01% formic acid-methanol (50:50) with isocratic elution for 4 min, a flow rate of 0.3 ml/min, and a column temperature of 30 °C [5].…”

“…Therefore, the derivatization procedure is carried out prior to analysis. Derivatization in this study was carried out using semicarbazide hydrochloride to form semicarbazone derivatives [5,14]. The procedure of derivatization was performed before the blood containing the analytes was applied on the DBS paper.…”

“…Quantitative analytical methods of cyclophosphamide and 4-OHCP have been described previously using High-Performance Liquid Chromatography (HPLC) [4] and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) [5][6][7]. However, quantitative analytical methods of cyclophosphamide and 4-OHCP in DBS simultaneously using UPLC-MS/MS have never been done before.…”

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.…”