Impurity profiling of alfentanil hydrochloride by liquid chromatography/quadrupole time-of-flight high-resolution mass spectrometric techniques for drug enforcement.

RATIONALE: Fentanyl and its analogues play important roles in the hospital and clinic setting as anesthetics. However, illicitly manufactured fentanyl as well as the new psychoactive substances (NPS) account for 30% of all deaths in the United States. Since fentanyl derivatives and NPS are designed to produce similar effects, their related substances are similar or even have the same active groups. A comprehensive analysis of the related substances of alfentanil hydrochloride can provide a basis for the identification and supervision of fentanyl derivatives and NPS. METHODS: A liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/QTOF-MS/MS) method was developed for the separation and characterization of related substances in alfentanil hydrochloride. Degradation studies were conducted according to the ICH-prescribed stress conditions. The compounds were identified mainly through positive electrospray ionization QTOF high-resolution mass spectrometric measurements of the accurate masses of the precursor and product ions and their calculated elemental compositions. Their formation mechanisms were also discussed. RESULTS: Seventeen related substances were detected in alfentanil hydrochloride and its stressed samples. Among them, nine were process-related substances and the other eight were degradation products. The stress study results demonstrated that alfentanil hydrochloride was unstable under acid, alkaline, and oxidative stress conditions, while relatively stable under dry photolytic and thermal stress conditions. Alfentanil hydrochloride was most susceptible for degradation at the N-phenylpropanamide and piperidine sites. CONCLUSIONS: Process-related alfentanil hydrochloride compounds are useful for determination of synthetic routes and entangling of fentanyl analogues. The stress study results can provide a sound scientific basis for the waste water monitoring of alfentanil. These results are important for routine quality control in the manufacturing and storage of alfentanil hydrochloride, as well as for drug enforcement of fentanyl and its analogues.

Data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS.

Recently, fentanyl analogs account for significant number of opioid deaths in the United States. Routine forensic analyses are often unable to detect and differentiate these analogs due to low concentrations and presence of structural isomers. A data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid was developed and validated using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Data were acquired using Time of Flight (TOF) and All Ions Fragmentation (AIF) modes. The limits of detection (LOD) in blood were 0.1-1.0 ng/mL and 0.1-1.0 ng/mL in TOF and AIF modes, respectively. In oral fluid, the LODs were 0.25 ng/mL and 0.25-2.5 ng/mL in TOF and AIF modes, respectively. Matrix effects in blood were acceptable for most analytes (1-14.4%), while the nor-metabolites exhibited ion suppression >25%. Matrix effects in oral fluid were -11.7 to 13.3%. Stability was assessed after 24 h in the autosampler (4 °C) and refrigerator (4 °C). Processed blood and oral fluid samples were considered stable with -14.6 to 4.6% and -10.1 to 2.3% bias, respectively. For refrigerated stability, bias was -23.3 to 8.2% (blood) and -20.1 to 20.0% (oral fluid). Remifentanil exhibited >20% loss in both matrices. For proof of applicability, postmortem blood (n = 30) and oral fluid samples (n = 20) were analyzed. As a result, six fentanyl analogs were detected in the blood samples with furanyl fentanyl and 4-ANPP being the most prevalent. No fentanyl analogs were detected in the oral fluid samples. This study presents a validated screening technique for fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS with low limits of detection.

Application of a screening method for fentanyl and its analogues using UHPLC-QTOF-MS with data-independent acquisition (DIA) in MSE mode and retrospective analysis of authentic forensic blood samples.

The steady appearance of new fentanyl analogues and the associated overdose deaths require the development of sensitive screening approaches to detect these compounds in biological samples and seizures. We developed a targeted screening method to detect 50 4-anilidopiperidine-related fentanyl analogues in whole blood using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry in data-independent acquisition mode. Sample preparation was performed using protein precipitation on a fully automated robotic setup. Thirteen analogues were selected to validate the method. A small matrix ion enhancement effect (110-123%) was observed for all of the compounds; the recovery ranged from 67% to 81% and the process efficiency from 81% to 98%. Limit of detection was within 0.0005-0.001 mg/kg and limit of identification ranged from 0.001 to 0.005 mg/kg. In the retrospective analysis of 2339 forensic blood samples, the major finding was fentanyl (n = 56), followed by alfentanil (n = 5) and remifentanil (n = 1). Identification of 34 fentanyl analogues was based on the predicted product ions resulting from common fentanyl-specific collision-induced cleavages, particularly on the product ion result of the fragmentation on the C-N bond between the phenylamide moiety and the piperidine ring. The proposed hypothesis was supported by the targeted analysis of 16 fentanyl analogues using this method and available published mass spectral data sources for fentanyl analogues. A targeted screening method for 50 fentanyl analogues was successfully validated and implemented to analyse authentic blood samples, where identifying targeted fentanyl analogues was tentatively achieved without using reference standards.

Surfactant assisted pulsed two-phase electromembrane extraction followed by GC analysis for quantification of basic drugs in biological samples.

In this work, a simple and efficient surfactant assisted pulsed two-phase electromembrane extraction (SA-PEME) procedure combined with gas chromatography (GC) has been developed for the determination of alfentanil, sufentanil and methadone in various samples. It has been found that the addition of anionic surfactant causes the accumulation of the cationic analytes at the SLM/solution interface resulting in an easier transfer of the analytes into the organic phase. The method was accomplished with 1-octanol as the acceptor phase and supported liquid membrane (SLM) by means of an 80 V pulsed electrical driving force and the extraction time of 20 min. The model analytes were extracted from 3.0 mL sample solution (pH 4.0) containing 0.02% w/v surfactant (sodium dodecyl sulfate). The duty cycle of 92% and frequency of 0.357 Hz gave the best performance. Extraction recoveries in the range of 70.5-95.2% and satisfactory repeatability (7.6

Miniaturized hollow fibre assisted liquid-phase microextraction and gas chromatography for determination of trace concentration of sufentanil and alfentanil in biological samples.

A simple and highly sensitive method that involves miniaturized hollow fibre assisted liquid-phase microextraction with gas chromatography-flame ionization detector was developed for the determination of trace concentration of sufentanil and alfentanil in biological samples. These drugs were extracted from 5 ml of aqueous solution with pH 10.0 into an organic extracting solvent (1-octanol) impregnated in the pores and lumen of a hollow fibre. After extraction for a prescribed time, 2.0 µl of the extraction solvent was injected directly in to the GC injection port. Under the optimized conditions, (1-octanol as extracting solvent, stirring rate of 700 rpm, 15% (w/v) salt addition, pH 10.0 and 25 min sampling time at 50 °C) large enrichment factors of 535 and 420 were achieved for sufentanil and alfentanil, respectively. Dynamic linear ranges were in the range of 0.05 to 500 ng/ml for sufentanil and 0.1 to 500 ng/ml for alfentanil. Limits of detection 0.01 and 0.02 ng/ml were obtained for sufentanil and alfentanil, respectively. The percent relative intra-day and inter-day standard deviations were found to be less than 8.4% (n = 5). Finally, this method was successfully applied for the separation, preconcentration and determination of trace concentration of sufentanil and alfentanil in plasma and urine samples.

Comparison of dispersive liquid-liquid microextraction and hollow fiber liquid-liquid-liquid microextraction for the determination of fentanyl, alfentanil, and sufentanil in water and biological fluids by high-performance liquid chromatography.

Dispersive liquid-liquid microextraction (DLLME) and hollow fiber liquid-liquid-liquid microextraction (HF-LLLME) combined with HPLC-DAD have been applied for the determination of three narcotic drugs (alfentanil, fentanyl, and sufentanil) in biological samples (human plasma and urine). Different DLLME parameters influencing the extraction efficiency such as type and volume of the extraction solvent and the disperser solvent, concentration of NaOH, and salt addition were investigated. In the HF-LLLME, the effects of important parameters including organic solvent type, concentration of NaOH as donor solution, concentration of H(2)SO(4) as acceptor phase, salt addition, stirring rate, temperature, and extraction time were investigated and optimized. The results showed that both extraction methods exhibited good linearity, precision, enrichment factor, and detection limit. Under optimal condition, the limits of detection ranged from 0.4 to 1.9 µg/L and from 1.1 to 2.3 µg/L for DLLME and HF-LLLME, respectively. For DLLME, the intra- and inter-day precisions were 1.7-6.4% and 14.2-15.9%, respectively; and for HF-LLLME were 0.7-5.2% and 3.3-10.1%, respectively. The enrichment factors were from 275 to 325 and 190 to 237 for DLLME and HF-LLLME, respectively. The applicability of the proposed methods was investigated by analyzing biological samples. For analysis of human plasma and urine samples, HF-LLLME showed higher precision, more effective sample clean-up, higher extraction efficiency, lower organic solvent consumption than DLLME.

Development and validation of a highly sensitive gas chromatographic-mass spectrometric screening method for the simultaneous determination of nanogram levels of fentanyl, sufentanil and alfentanil in air and surface contamination wipes.

A highly sensitive gas chromatographic-mass spectrometric (GC-MS) analytical method for the determination of the opioid narcotics fentanyl, alfentanil, and sufentanil in industrial hygiene personal air samples and surface contamination wipes was developed and comprehensively validated. Sample preparation involved a single step extraction of the samples with methanol, fortified with a fixed amount of the penta-deuterated analogues of the opioid narcotics as internal standard. The GC-MS analytical procedure using selected ion monitoring (SIM) was shown to be highly selective. Linearity was shown for levels of extracted wipe and air samples corresponding to at least 0.1-2 times their surface contamination limit (SCL) and accordingly to 0.1-2 times their time weighted average occupational exposure limit (OEL-TWA) based on a full shift 9601 air sample. Extraction recoveries were determined for spiked air samples and surface wipes and were found to be quantitative for both sampling media in the entire range studied. The air sampling method's limit of detection (LOD) was determined to be 0.4 ng per sample for fentanyl and sufentanil and 1.6 ng per sample for alfentanil, corresponding to less than 1% of their individual OEL for a full shift air sample (9601). The limit of quantification (LOQ) was found to be 1.4, 1.2, and 5.0 ng per filter for fentanyl, sufentanil, and alfentanil, respectively. The wipe sampling method had LODs of 4 ng per wipe for fentanyl and sufentanil and 16 ng per wipe for alfentanil and LOQs of respectively, 14, 12, and 50 ng per wipe. The analytical intra-assay precision of the air sampling and wipe sampling method, defined as the coefficient of variation on the analytical result of six replicate spiked media was below 10 and 5%, respectively, for all opioids at all spike levels. Accuracy expressed as relative error was determined to be below 10%, except for alfentanil at the lowest spike level (-13.1%). The stability of the opioids during simulated air sampling was investigated. For fentanyl and sufentanil a quantitative recovery was observed at all spike levels, while for alfentanil recoveries ranged from 60.3 to 85.4%. When spiked air samples were stored at ambient temperature and at -15 degrees C quantitative recovery was found for fentanyl and sufentanil after 7 and 14 days. For alfentanil a slight loss seemed to occur upon storage during 7 days, being more explicit after 14 days. Ambient storage of spiked wipes seemed to lead to significant losses of all opioids studied, yielding recoveries of 37.7-88.3%. Upon storage of similar wipes at -15 degrees C a significantly higher recovery was found ranging from 77.3 to 88.3%. The developed analytical and sampling procedures have been recently applied in an explorative field study of which the results of surface contamination wipe sampling are presented in this paper. To our knowledge, this is the first study addressing the development and validation of analytical procedures for the assessment of external occupational exposure to potent opioid narcotics.

Development and validation of an HPLC assay for fentanyl, alfentanil, and sufentanil in swab samples.

A high performance liquid chromatography (HPLC) method for the assay of fentanyl citrate, alfentanil hydrochloride, and sufentanil citrate swab samples was developed and validated in order to control a cleaning procedure. The swabbing procedure involved Super POLX 1200 wipers moistened with water. The assay employed extraction of swabs with water and analysis by isocratic, reversed-phase, HPLC with varying ultraviolet (UV) detection for desired sensitivity, depending on the analyte. The method was shown to be selective and linear from the limits of quantitation (0.10, 0.20, and 0.15 microg/swab for fentanyl citrate, alfentanil, and sufentanil, respectively) to over three times these concentrations. The assay limits (detection levels) per swab area were set at least at 0.2% of the concentrations of the actives in the drug products (0.02, 0.10, and 0.10 microg/swab or approximately 0.03, 0.02, and 0.2% for fentanyl citrate, alfentanil, and sufentanil, respectively). It should be noted that all active concentrations listed in this work were calculated based on the salt form concentration for fentanyl (citrate salt) and the free base forms for alfentanil and sufentanil. No reference standard was available for alfentanil hydrochloride and sufentanil citrate. Drug product was used instead throughout this study.

Cardiovascular effects of equipotent isoflurane and alfentanil/isoflurane minimum alveolar concentration multiple in cats.

OBJECTIVE: To determine whether administration of opioids to anesthetized cats induced less cardiovascular depression than that induced by an equivalent amount of anesthetic alone, and to measure endocrine responses to a noxious stimulus. ANIMALS: 6 healthy female cats. PROCEDURE: Anesthesia was induced with isoflurane and was maintained for 60 minutes at 1.3 isoflurane MAC. Blood gas tensions, pH, and plasma alfentanil and hormone concentrations, blood pressures, and cardiac output were measured. A noxious stimulus was applied for 5 minutes, while blood acquisition and measurements were repeated. Alfentanil was administered i.v. to achieve estimated plasma concentration of 500 ng/ml, and end-tidal isoflurane concentration was reduced by 35%. After another 60 minutes, blood was obtained and measurements were taken, then a second 5-minute noxious stimulus was applied while blood acquisition and measurements were retaken. RESULTS: Alfentanil administration and reduction of isoflurane concentration significantly increased body temperature, heart rate, mean arterial pressure, mean pulmonary arterial pressure, stroke index, cardiac index, hemoglobin, oxygen delivery index, PvO2 and PvCO2, dopamine, epinephrine (EPI), norepinephrine (NOREPI), and cortisol values, and significantly decreased arterial and venous pH. Application of a noxious stimulus significantly increased heart rate, stroke index, cardiac index, PaO2, oxygen delivery index, arterial and venous pH, and NOREPI values, and decreased bicarbonate, PaCO2, PvCO2, and EPI values. Alfentanil administration blunted cardiac index, PaCO2, oxygen delivery index, arterial pH, PaO2, and EPI, and NOREPI responses to a noxious stimulus. CONCLUSIONS: Compared with isoflurane alone, alfentanil administration and reduction of isoflurane MAC improved cardiovascular variables, and blunted respiratory, hormonal, and most hemodynamic responses to a noxious stimulus in cats. CLINICAL RELEVANCE: Use of the balanced opioid anesthesia regimen induced some beneficial effects in healthy cats; effects were similar to, although greater in nature, than effects induced by a noxious stimulus.

Gas chromatographic-mass spectrometric analysis of alfentanil metabolites. Application to human liver microsomal alfentanil biotransformation.

The short-acting synthetic opioid alfentanil undergoes extensive biotransformation to several metabolites. A gas chromatographic-mass spectrometric assay, using selected-ion monitoring and deuterated internal standards, was developed for quantitating the predominant metabolites of alfentanil. Optimal extraction and derivatization conditions are described. The assay was applied to the analysis of metabolites formed during alfentanil metabolism in vitro by human liver microsomes. Formation of known alfentanil metabolites was confirmed, and formation of a metabolite, not previously detected in vitro, is described. The assay represents a significant improvement over existing methods of alfentanil metabolite analysis, which use HPLC and radiochemical detection.

Use of refractometry to identify opioid-containing solutions.

The purpose of this laboratory study was to assess the value of refractometry in identifying the contents of a variety of opioid-containing solutions. A hand-held refractometer was used to document the refraction produced by the undiluted contents of alfentanil, fentanyl, morphine, sufentanil ampoules and by solutions of Ringer's lactate, 0.9% saline, 3.3% dextrose in 0.3% saline, and distilled water. Each opioid was then serially diluted in serial 1:2, 1:4, and 1:8 dilutions in each of these solutions and the refractions of each determined. Based on this information, blinded identification of various diluted opioid solutions was attempted. Refractometer values for undiluted fentanyl and sufentanil were identical with those for distilled water. Those for undiluted alfentanil and morphine were almost identical with each other and with 1:2 and 1:4 dilutions of either drug in Ringer's lactate or 0.9% saline. We conclude that refractometry is an unreliable screening method to detect tampering with opioid solutions.

Safety and efficacy of alfentanil and halothane in paediatric surgical patients.

Alfentanil, a congener of the opioid fentanyl, possesses properties that make it an attractive choice for use during short operative procedures. Since the pharmacodynamic aspects of alfentanil have not been well documented in children, this study was undertaken to evaluate the safety, efficacy, and dose requirements of alfentanil when used with nitrous oxide or halothane in paediatric patients. Eighty unpremedicated patients, ASA physical status I or II and aged 2-12 yr were studied. Patients were randomly assigned to one of four groups. After induction of anaesthesia with nitrous oxide, oxygen, and halothane, the groups were treated as follows. In Group 1 (n = 19), after halothane was discontinued, alfentanil 50 micrograms.kg-1 was infused over 30 sec. In Group 2 (n = 20), the end-tidal halothan was maintained at 0.5% and alfentanil 25 micrograms.kg-1 was infused. In Group 3 (n = 20), the end-tidal halothane concentration was maintained at 1% and alfentanil 12.5 micrograms.kg-1 was infused. In Group 4 (n = 21), the end-tidal halothane concentration was maintained at 1.5% and no alfentanil was administered. Patients in Groups 1, 2, and 3 received bolus doses of alfentanil 12.5 micrograms.kg-1 as needed to maintain haemodynamic stability. After alfentanil administration, there were transient decreases in systolic blood pressure in Groups 1 and 2, and in heart rate in Group 2. With surgical stimulation, haemodynamic stability was well maintained except in patients in Group 1, who had an increase in systolic blood pressure. Children Group 1 were alert sooner and their tracheas were extubated earlier than those in Groups 2, 3, and 4.(ABSTRACT TRUNCATED AT 250 WORDS)