LC-MS/MS quantification of ropivacaine and local analgesic and adverse effects of Long-acting Ropivacaine Injection based on pharmacokinetic-pharmacodynamic modelling in Bama minipigs.

The local analgesic efficacy and adverse effects of a new Long-acting Ropivacaine formulation were examined based on pharmacokinetic-pharmacodynamic (PK-PD) modelling in Bama minipigs. 24 Bama minipigs, 12 males and 12 females, were randomly and equally divided into the following treatment groups: normal saline injection, drug vehicle injection, Long-acting Ropivacaine Injection and Ropivacaine Hydrochloride Injection. After routine disinfection, a skin incision about 3 cm long and 3 cm deep was produced in the leg of each pig, and mechanical withdrawal threshold (MWT) measured at various times pre- and post-injection as an index of analgesia against incision pain. Plasma ropivacaine concentrations were also measured at the same times using a novel liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method. Minipigs were sacrificed 24 h post-injection and hearts collected for drug concentration measurements by LC-MS/MS. The LC-MS/MS method demonstrated high sensitivity, linearity and precision. The Long-acting Ropivacaine formulation produced a longer analgesic effect (∼12 h) at a lower plasma concentration than Ropivacaine Hydrochloride (∼4h), suggesting a better side-effects profile. A PK-PD model revealed a direct relationship between plasma ropivacaine concentration and MWT, with peak analgesia at about 1000 ng/mL and behaved good prediction ability. Long-acting Ropivacaine Injection is a superior local anaesthetic-analgesic treatment due to longer-lasting efficacy at lower concentrations compared to Ropivacaine Hydrochloride, which will reduce the risk of side effects such as cardiotoxicity.

Determination of Fentanyl, Alpha-Methylfentanyl, Beta-Hydroxyfentanyl and the Metabolite Norfentanyl in Rat Urine by LC-MS-MS.

Fentanyl and its analogs are potent synthetic opioids with a high potential for abuse and dependence. They have become major contributors to opioid deaths. This study aimed to determine whether the metabolites of fentanyl, alpha-methylfentanyl and beta-hydroxyfentanyl, excreted in the urine, can demonstrate historical drug exposure. Fentanyl is primarily metabolized via CYP3A4 into norfentanyl, although there is little research on its metabolization into alpha-methylfentanyl and beta-hydroxyfentanyl. We conducted in vitro experiments with human liver microsomes (HLMs) and rat liver microsomes (RLMs) to elucidate the major metabolic pathways of alpha-methylfentanyl and beta-hydroxyfentanyl using ultra-high-performance liquid chromatography coupled with mass spectrometry. The results showed that both alpha-methylfentanyl and beta-hydroxyfentanyl were predominantly metabolized into norfentanyl in HLM and RLM. Urine samples were collected at different intervals from 0 h to 72 h after intravenous administration of alpha-methylfentanyl and beta-hydroxyfentanyl (20 µg/kg) to Sprague-Dawley rats. We prepared the samples by liquid-liquid extraction, and the internal standard (IS) was cariprazine. A sensitive, rapid liquid chromatography-tandem mass spectrometry method was developed and validated to determine four analytes in the urine. The lower limit of qualification in urine was 2 pg/mL for fentanyl, 5 pg/mL for alpha-methylfentanyl, 10 pg/mL for beta-hydroxyfentanyl and 40 pg/mL for norfentanyl. The analytical range was 0.002-2 ng/mL for fentanyl, 0.005-5 ng/mL for alpha-methylfentanyl, 0.01-10 ng/mL for beta-hydroxyfentanyl and 0.04-40 ng/mL for norfentanyl. All analytes demonstrated good linearity (R2 > 0.99). The extraction recoveries were in the 67.8%-92.1% range, and the IS-normalized matrix effects were between 55.5% and 74.0% (coefficient of variance < 15%). Our data indicated that norfentanyl has a higher concentration in rat urine and was detectable for at least 3 days after exposure to these compounds. This developed method may be useful in various fields, including forensic analysis, workplace drug testing and monitoring drug abuse.

Alpha-Methylfentanyl and Beta-Hydroxyfentanyl LC-MS-MS Quantification in Rat Plasma after Long-Term Ethanol Exposure.

Fentanyl and its analogues are highly abused drugs that dominate the illicit drug trade. alpha-Methylfentanyl (A-F) and beta-hydroxyfentanyl (B-F) are two fentanyl analogues that require the development of rapid detection technologies. The current study established and validated a rapid and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS-MS) method to measure A-F and B-F concentrations in rat plasma following intravenous drug administration (20 µg/kg). Because fentanyl is primarily metabolized by the liver, we evaluated the concentrations of A-F and B-F in vivo in rats, in a control group and a group with liver damage induced by 55 days of oral ethanol gavage (6.5 g/kg, 22.5% v/v). Liquid-liquid extraction and LC-MS-MS operating in the positive ion multiple reaction monitoring mode were used. A C18 column was used, and the mobile phase consisted of 0.1% formic acid aqueous and acetonitrile. The limit of detection was 3 pg/mL (S/N > 5) for A-F and B-F. The calibration curves were linear within the concentration range of 0.01-5 ng/mL (R2 = 0.9991) and 0.005-20 ng/mL (R2 = 0.9999) for A-F and B-F, respectively. Extraction recoveries were 91.3%-97.6% with RSD ≤ 11.2% and 90.5%-94.3% with RSD ≤ 10.5% for A-F and B-F, respectively. Plasma matrix effects were 80.61%-84.58% for A-F and 80.67%-81.33% for B-F with RSD ≤ 13.9%. The validated assay indicated no significant differences in pharmacokinetic parameters (AUC0-t, Cmax and T1/2) derived from the assessment of A-F and B-F plasma concentrations between control and ethanol-exposed rats. This assay, for which the LOD was 3 pg/mL for A-F and B-F may help the forensic science field to determine fentanyl analogue-related causes of death and identify illicit drug tampering.