Abstract
A fast, accurate and reliable ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.
Highlights
Malaria, a vector-borne disease caused by the Plasmodium parasite, continues to have a devastating impact on people’s health and lives around the world [1]
The simplicity and suitability of the procedure for large sample set analysis were taken into consideration during the ultra-high performance liquid chromatograph (UHPLC)-MS/MS method development and optimization
The mean found concentration fell within the acceptance ranges for accuracy and precision for all analytes at the tested concentration levels (5 and 50 ng/mL, see Table 4). These results demonstrate that analyte concentrations were not significantly affected by the applied extraction method, showing the practicability of the method for quantification of the analytes in bovine plasma
Summary
A vector-borne disease caused by the Plasmodium parasite, continues to have a devastating impact on people’s health and lives around the world [1]. Zoo-prophylaxis of bovines with Macrocyclic Lactones (MLs) against An. Zoo-prophylaxis of bovines with Macrocyclic Lactones (MLs) against An Arabiensis, as it preferentially feeds on cattle, is a novel and complementary approach for malaria control and elimination, especially in East and Central Africa [7,8,9,10,11]. The avermectins (ivermectin (IVER) and doramectin (DORA)) and milbemycins (moxidectin (MOXI)) are 6-membered MLs endectocides commonly used against veterinary helminths, ectoparasites. They are semi-synthetic fermentation products of soil-dwelling bacteria of the genus Streptomyces with an excellent safety profile [12]. The use of MLs has been extended to humans, especially IVER, against onchocerciasis (river blindness) and is a candidate drug for malaria vector control when given to cattle targeting An. Arabiensis [14]. Restrictions usually apply to ship samples from endemic regions to other countries based on the possible transmission of infectious agents, such as foot-and-mouth disease (FMD)
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