Development and validation of an LC–MS/MS method for the simultaneous quantification of milbemycin oxime and praziquantel in plasma: application to a pharmacokinetic study in cats

Introduction Milbemycin oxime (MBO) and praziquantel (PZQ) have a broad spectrum of biological activity and are commonly used to treat the parasitic infection in the veterinary clinic. In this study, a fast and efficient LC-MS/MS method was established and validated for the simultaneous determination of MBO, PZQ, cis-4-hydroxylated-PZQ (C-4-OH-PZQ) and trans-4-hydroxylated-PZQ (T-4-OH-PZQ) and in cat plasma. Methods Extraction of analytes and internal standards from cat plasma by acetonitrile protein precipitation, allows rapid processing of large batches of samples. MBO, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, and internal standard (IS) were eluted for 13.5 min on a C18 column with a 0.1% formic acid water/acetonitrile mixture as the mobile phase. Results Results showed that the method had good precision, accuracy, recovery, and linearity. The linearity range was 2.5–250 ng/mL for MBO, and 10–1000 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ. The intra-day and inter-day precision CV values of the tested components were within 15%. The extraction recoveries of the four components ranged from 98.09% to 107.46%. The analytes in plasma remained stable for 6 h at room temperature, 26 h in the autosampler (4 °C), after freeze–thaw (−20°C) cycles, and 60 days in a −20°C freezer. Method sensitivity sufficed for assessing pharmacokinetic parameters of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in plasma samples with LLOQ of 2.5 ng/mL for MBO and 10 ng/mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ. Conclusion In this study, a selective and sensitive LC-MS/MS method for the simultaneous quantification of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in cat plasma was developed and validated.This method had been successfully applied to evaluate the pharmacokinetics of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ after a single oral administration of 8 mg MBO and 20 mg PZQ in cats.


Introduction
Praziquantel (PZQ), a broad-spectrum thiazolone-pyrazineisoquinoline antiparasitic derivative, is safe for the treatment of parasitic infections in cattle and is also used for the treatment of schistosomiasis in humans (1,2).PZQ is the only drug widely used in schistosomiasis control programs worldwide because it is effective, inexpensive, and easy to administer in one dose (3).PZQ is a racemic mixture composed of equal amounts of the R (−) isomer levo-PZQ and the S (+) isomer dextro-PZQ (4).The R (−) enantiomer has antischistosomal activity, but the (S) enantiomer does not have antischistosomal action (5).R-PZQ is primarily metabolized to R-cis-4-OH-PZQ, whereas S-PZQ is broken down to various mono-and di-hydroxy metabolites in addition to S-trans-and S-cis-4-OH-PZQ (6).PZQ has been used in combination with other various compounds, such as pyrantel pamoate, febantel, and milbemycin oxime, to create broad-spectrum insect repellents for pets (7)(8)(9).
Milbemycin oxime (MBO) is a macrocyclic lactone isolated from Streptomyces, consisting of nitrile derivatives of 5-didehydromilbemycin, with the proportion of 80% A4 milbemycin (C 32 H 45 NO 7 , MW 555.71) and 20% A3 milbemycin (C 31 H 43 NO 7 , MW541.68) (10).MBO is structurally and chemically similar to avermectin; it exhibits similar anthelmintic, insecticidal, and acaricidal effects at low dose, and is less toxic to mammals (11).MBO is active against the larval and adult stages of intestinal nematodes and the blood stage of heartworm larvae (12).Companion animals may be exposed to infections from internal and external parasites (13).MBO has recently been used in combination with PZQ to develop new treatments against cestodes, nematodes, and ectoparasites in companion animals (14,15).MBO/PZQ chewable tablets was approved by US-FDA for the prevention of heartworm disease caused by Dirofilaria immitis and for the treatment and control of adult roundworm (Toxocara canis, Toxascaris leonina), adult hookworm (Ancylostoma caninum), adult whipworm (Trichuris vulpis), and adult tapeworm (Taenia pisiformis, Echinococcus multilocularis, and Echinococcus granulosus) infections (16).The utilization of a combination of MBO and PZQ for cats, exemplified by Milpro ® (Virbac, France) and Milbemax ® (Novartis, France), has obtained approval (15,17).
Previous methods have been developed for the analysis of MBO, and PZQ separately (18)(19)(20).Few studies have been reported for the simultaneous quantitation of MBO, PZQ, and its main metabolite.The objective of this study was to develop a sensitive, reproducible and efficient LC-MS/MS method to simultaneously quantify MBO, PZQ, and its main metabolite (cis-4-hydroxylated-PZQ and trans-4hydroxylated-PZQ).The developed method has been fully validated and successfully applied to the pharmacokinetic study of MBO and PZQ receiving oral administration in cats.This method will facilitate the study of bioequivalence.

Sample preparation
A total of 200 μL of cat plasma was extracted with a mixture of 800 μL acetonitrile containing IS.The tube was then agitated on vortex mixer for 30 s ans centrifuged at 12000 rpm for 10 min at 4°C.The 0.5 mL supernatant was added 0.5 mL ultrapure water filtered through a 0.22 μm nylon syringe filter and transferred to microvials for analysis.

Instrument and conditions
The LC-MS/MS system consisted of a Nexera XR HPLC system (Shimadzu, Japan) coupled with an LCMS-8050 triple quadrupole MS (Shimadzu, Japan).LabSolutions 5.99 software was used for data collection and processing.All experiments were performed in positive ion mode using an electrospray ionization (ESI) source.Table 1 shows the MRM transitions and other MS parameters for all analyte in this method.
The chromatographic column was Gemini C 18 (50 mm × 2.0 mm; 5 μm; Phenomenex), the solvents were 0.1% formic acid (solvent A) and acetonitrile (solvent B), and the gradient elution rate was 0.3 mL/ min.Gradient conditions were optimized as follows: 15-30% B at 0-0.5 min, 30% B at 0.5-2.5 min, 30-50% B at 2.5-2.6 min, 50% B at 2.6-4.5 min, 50-85% B at 4.5-6.0min, 85% B at 6.0-10.5 min, 85-15% B at 10.5-11.0min, and 15% B at 11.0-13.5 min.The injection volume was 10 μL.The sample chamber temperature of the autosampler was set to 4°C, and the column temperature was set to 35°C.The standard curve samples were prepared according to the protocols described in Section 2.2.The internal standard calibration curves plot the ratio of the analyte response to the internal standard response (response factor) against the ratio of the analyte amount to the internal standard amount.A correlation coefficient (R 2 ) of 0.99 or above was considered suitable.

Accuracy and precision
Assay accuracy and precision were measured at four different concentrations (2.5, 7.5, 30, and 200 ng/mL of MBO; 10, 30, 120, and 800 ng/mL of PZQ, C-4-OH-PZQ, and T-4-OH-PZQ).Accuracy and precision were determined between runs (intraassay) and recorded on three different days (inter-assay).The precision of the method was assessed using the coefficient of variation (CV), with a CV ≤ 15% being an acceptable value.Accuracy was expressed as a percentage of the measured QC concentration relative to the nominal value.

Recovery and matrix effect
Absolute recoveries of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ were assessed using the calculated ratio of blank plasma; the concentration spiked into QC samples to its nominal concentration.Matrix effects were assessed by comparing the peak areas of the extracted samples with those of pure solutions of the same concentration.

Pharmacokinetics
All animal procedures were approved by the Institutional Animal Care and Use Committee of South China Agricultural University (approval number: 2023A011), and the cats were handled with due regard for their welfare and in compliance with all local and national regulatory requirements.Six male cats were studied to determine the PK profiles of MBO, PZQ and its main metabolite (C-4-OH-PZQ and T-4-OH-PZQ).The cats were ≥ 12 months of age and weighed between 2.7 and 3.8 kg.The cats were fasted overnight and allowed free access to water prior to PK study.A single dose of 8 mg MBO and 20 mg PZQ formulation was administered to cats. 1 mL of blood samples were drawn from the antecubital vein and collected in tubes containing heparin at the following times: 0, 0.16, 0.5, 0.75,1, 2,3, 4, 6, 9, 12, 16, 24, 36, 48, 72, 120, 168, and 216 h.Blood samples were centrifuged at 3500 rpm for 10 min at 4°C to separate plasma and stored at −20°C until analysis.The plasma PK parameters of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ were determined by non-compartmental analysis in Phoenix WinNonlin ® 8.2 (Certara, L.P., Princeton, NJ, United States).To avoid cross-peaks, gradient elution was used to separate the different analytes on a C 18 column.Methanol, acetonitrile, 5 mM ammonia acetate (pH = 7.6), 0.1% formic acid, and 100% water were used as potential mobile phases.A gradient elution was established as described in section 2.4.The results showed that when eluted with 0.1% formic acid and acetonitrile, the peak resolution and peak shape are better.Under the optimized conditions, MBO-A3, MBO-A4, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, IS1, and IS2 were separated on a C 18 column eluted with 0.1% formic acid and acetonitrile under gradient conditions (13.5 min).These conditions allowed for high chromatographic resolution with clear MBO-A3, MBO-A4, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, IS1 and IS2 peaks at 8.1, 8.5, 4.8, 2.7, 2.4, 8.6, and 4.8 min, respectively.

Selection of IS
The use of internal standards can substantially reduce the random errors caused by the extraction process, ionization, and injection (21).To obtain better accuracy and precision, abamectin and moxidectin were tested as IS for MBO.Due to the instability of abamectin, moxidectin was finally selected as IS for MBO because it not only reduces the influence of the matrix on the analyte, but also more effectively reduced in the influence of the volume changes during the sample extraction.PZQ-d11 was finally selected as IS for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ (22).

Sample preparation
Some of the most commonly used sample preparation methods for the simultaneous purification of plasma samples and the extraction of drugs include protein precipitation (23), liquid-liquid extraction (24), solid phase extraction (25), and their combinations (26).In this study, the one-step protein precipitation method was ultimately selected given its rapid and simple performance.During method development, we used methanol and acetonitrile as solvents for protein precipitation, and ultimately selected acetonitrile because of its extended recovery yield.The pretreatment method can analyze a large number of plasma samples in a short time.

Method validation 3.4.1. Selectivity
The chromatograms of blank plasma, blank plasma spiked with MBO, PZQ, C-4-OH-PZQ, T-4-OH-PZQ, IS1, and IS2, and a PK sample at 3 h are shown in Figure 2. Comparing these chromatograms, no endogenous interferences were detected in the retention times of the analyte and IS.Therefore, the plasma background does not interfere with MBO, PZQ, C-4-OH-PZQ, T-4-OH-PZQ and IS, suggesting the selectivity and specificity of the MRM.

Plasma stability
Stability tests showed that MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ were stable in three freeze-thaw cycles, for 6 h at room temperature, after processing for 26 h at 4°C, and storage (−20°C) condition (Table 5).Four stability tests obtained an accuracy between 93.00 and 111.07%and an RSD% ≤10% for all analytes.All analytes were considered storage stable according to FDA criteria (28) with an accuracy of ±15% at each level under all conditions tested.

Dilution integrity
When plasma sample concentrations exceed the ULOQ, a fivefold dilution was performed to quantify MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in the cat plasma samples within the range of the standard curve.Analytes were diluted five-fold with empty cat plasma to QC concentrations of 200 ng/mL (MBO) and 800 ng/mL (PZQ, C-4-OH-PZQ, and T-4-OH-PZQ), and plasma samples were collected in duplicate six times to check the completeness of the dilution.Table 6 represents the results of the five-fold dilution validation experiment of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in cat plasma.The accuracy and precision of the diluted concentrations satisfied the acceptance criteria, defined as ±15% of the nominal concentrations, indicating that the bioanalytical method is valid for dilution integrity of samples at concentrations exceeding the ULOQ.

Application in a PK study is cats
The mean MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ plasma concentration-time profiles after oral administration of 8 mg MBO and 20 mg PZQ to cats (n = 6) are shown in Figure 3.The PK parameters are listed in Table 7.The mean AUC 0 − t values of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ were 4820.76,3593.22,PZQ is the drug of choice for the treatment of schistosomiasis and is widely used in preventive chemotherapy programs (as defined by WHO) (29).PZQ is metabolized by multiple CYPs, and so other drugs within these CYP pathways may lead to the formation and accumulation of metabolic by-products or a reduce the drug's therapeutic efficacy of the drug (30).Although the structure and properties of MBO are very close to ivermectin, it is safer and has fewer side effects (31).There are a variety of compound antiparasitic products for pets.The impact of different combination of antiparasitic drugs on their respective pharmacokinetics requires further study (32).The LC-MS/MS method established in this study provides a practical means for the quantitative analysis of MBO and PZQ and metabolites.In addition, this method can also be applied to a bioequivalence studies.

Conclusion
In this study, a selective and sensitive LC-MS/MS method for the simultaneous quantification of MBO, PZQ, C-4-OH-PZQ, and T-4-OH-PZQ in cat plasma was developed and validated.The short run time (13.5 min) simplicity and reproducibility of the extraction method are valuable advantages for the analysis of a large number of samples.The sensitivity in plasma was 2.5 ng/mL for MBO and 10 ng/ mL for PZQ, C-4-OH-PZQ, and T-4-OH-PZQ.The applicability of this method was demonstrated by analyzing the PK profile of cats after a single oral dose of 8 mg MBO and 20 mg PZQ.

TABLE 1
Mass spectrometric conditions for each analyte.