High sensitivity method validated to quantify estradiol in human plasma by LC-MS/MS.

17ß-Estradiol (E2) is an endogenous steroid in the human body. Its measurement is important for health and human biology understanding. However, E2 concentration in human plasma is in the range of pg/mL, which makes it difficult to detect. In this way, LC-MS/MS has been shown the most sensitive tool, although E2 is a weakly ionizable molecule. In this work, we validated a more sensitive and accurate method for E2 quantification in human plasma. Our extraction step ensured a cleaner chromatography, resulting in a precise measurement and highly reproducible method in the range of 2-150pg/mL. Moreover, we proved a long stability for E2 in several conditions. All results indicate that our developed method is robust and sensitive enough to apply in bioequivalence studies for E2 measurement in human plasma, even at very low concentrations.

Pharmacokinetic and pharmacodynamic evaluation of a nanotechnological topical formulation of lidocaine/prilocaine (nanorap) in healthy volunteers.

BACKGROUND: Nanorap is a new nanotechnological formulation for topical anesthesia composed of lidocaine (2.5%) and prilocaine (2.5%). This study evaluated the pharmacokinetics of Nanorap. For the determination of lidocaine and prilocaine in human plasma, a new method using high-performance liquid chromatography coupled with tandem mass spectrometry was developed. Nanorap pharmacodynamic (PD) and its physical proprieties were also evaluated. METHODS: Nanorap was administered by topical application of 2 g to healthy volunteers, and blood samples were collected for the pharmacokinetics analysis. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, vol/vol). The chromatography separation was performed on a Genesis C18 analytical column 4 µm (100 × 2.1 mm i.d.) with a mobile phase of methanol/acetonitrile/water (40/30/30, for lidocaine, and 50/30/20, for prilocaine, vol/vol/vol) + 2 mM of ammonium acetate and ropivacaine as internal standard. The drugs were quantified using a mass spectrometer with an electrospray source in the electrospray ionization positive mode configured for multiple reaction monitoring. The PD of Nanorap was evaluated with the use of a visual analog scale. Nanorap was characterized by cryofracture. RESULTS: The chromatography run-time was 5.5 minutes for lidocaine and 3.3 minutes for prilocaine, and the lower limit of quantification was 0.05 ng/mL for both drugs. Mean Cmax was 6.62 and 1.72 ng/mL for lidocaine and prilocaine, respectively. Median Tmax was 6.5 hours for both drugs. Nanocapsules had a mean size of 88 nm and mean drug association of 92.5% and 89% for lidocaine and prilocaine, respectively. The PD study showed that Nanorap has a sufficient analgesic effect (>30% reduction in pain) after 10 minutes of application. CONCLUSIONS: A new simple, selective, and sensitive method for determination of lidocaine and prilocaine in human plasma was developed. Nanorap generated safe plasma levels of the drugs and satisfactory analgesic effect.

Tumescent local anesthesia with ropivacaine in different concentrations in bitches undergoing mastectomy: plasma concentration and post-operative analgesia.

OBJECTIVE: To compare two concentrations of ropivacaine administered for tumescent local anesthesia (TLA) in dogs undergoing mastectomy. STUDY DESIGN: Prospective randomized clinical study. ANIMALS: Seventeen bitches of various breeds, aged 12 ± 2 years and weighing 10 ± 6.5 kg requiring total unilateral or bilateral mastectomy. METHODS: Dogs were premedicated with acepromazine (0.04 mg kg(-1) ) and morphine (0.4 mg kg(-1) ) intramuscularly. Anesthesia was induced with propofol (2.5 mg kg(-1) ) and midazolam (0.2 mg kg(-1) ) intravenously, followed by intubation and maintenance with isoflurane and TLA. Dogs were randomly allocated to receive TLA either with 0.1% ropivacaine (group G1) or with 0.05% ropivacaine (group G05). TLA was performed by insertion of a multihole needle under the skin and infusion of ropivacaine and lactated Ringer's solution at a fixed volume of 15 mL kg(-1) . Ropivacaine concentrations in arterial blood were measured by high-performance liquid chromatography. Post-operative pain was assessed using two scales (University of Melbourne pain scale and a modified composite measure pain scale) and von Frey filaments, 4 hours after TLA and at 1 hour intervals until sensitivity was regained. A score above 30% of the maximum possible score was considered a positive indicator of pain. RESULTS: Peak plasma concentrations of ropivacaine were measured 240 minutes after TLA in G1. Low concentrations were measured in G05 for 60 minutes, with subsequent increase. Analgesic rescue and return of sensitivity occurred at 7 ± 2.3 and 7 ± 1.9 hours (mean ± SD) after TLA for G1 and G05, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Tumescent local anesthesia with ropivacaine provided satisfactory post-operative analgesia that lasted for several hours, with no difference in duration between the concentrations. No serious side effects were attributed to TLA. Results indicated that 0.05% ropivacaine provided adequate analgesia for mastectomy, however, more studies are required to support this conclusion.

Quantification of dapaconazole in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry: application to a phase I study.

A simple, selective and sensitive method based on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) has been developed for the determination of dapaconazole in human plasma using tioconazole as internal standard. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis(®) C18 reversed phase analytical column 4µm (100×2.1mm i.d.) with a mobile phase of methanol/acetonitrile/water (80/10/10, v/v/v)+ammonium acetate (0.5mM). Dapaconazole was quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring (MRM) to monitor the transitions 415.1>159.2 and 387.0>131.0 for dapaconazole and tioconazole, respectively. The method had a chromatography run time of 3.8min and a linear calibration curve over the range 0.2-100ng/mL (r=0.9998). The lower limit of quantification (LLOQ) was 0.2ng/mL. The precision and accuracy values of the assay were within ±10%. The stability tests indicate no significant degradation under the conditions of the experiment. This method was used for a phase I study of topical administration of dapaconazole tosylate in healthy human male volunteers.