A graphene tip coupled with liquid chromatography tandem mass spectrometry for the determination of four synthetic adulterants in slimming supplements.

Slimming supplements were popularly sold online driven by the increasement of obesity and the development of social networking platform. However, events of drug abuse in slimming supplements were also frequently reported. In this study, a graphene tip solid-phase extraction (Gtip SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for determining fenfluramine, phenolphthalein, bumetanide, and sibutramine in slimming supplements. It was validated in terms of linearity (0.9985-0.9995), LOD (1.8ngmL-1), LOQ (5.6ngmL-1), intra-day precision (<5.1%), inter-day precision (<7.3%), and recovery (82.9-95.2%). Sibutramine is the most commonly used drug, which was detected in Bihais, Galong, and Aolist, with content 12.4, 3.6, 20.3mgg-1, respectively. Phenolphthalein was also found with content lower than 5.2mgg-1. The successful application of Gtip SPE and UPLC-MS/MS method indicated its advantage in analyzing low level of contaminates resulted from violation of regulation.

[Analysis and identification of illegal constituents in health food products implicitly advertizing tonic or slimming effect in the National Institute of Health Sciences in Japan].

With the prefectural governments' aid of the purchase, the Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences (NIHS) successively has surveyed illegal constituents in health food products implicitly advertizing tonic or slimming effect since the fiscal year of 2002 (slimming type) or 2003 (tonic type). The average numbers of the analyzed products per year are about 100 (slimming type) and 150 (tonic type), respectively. We also continuously distribute standards of authentic samples of several illegal components such as N-nitrosofenfluramine (NFF) and sildenafil (SIL) to prefectural institutes and the average gross number per year is about 140. In the case of slimming type, the fact that the products containing NFF were widely sold in Japanese markets in 2002 is well known. In addition, phenolphthalein, fenfluramine, sibtramine, desdimethylsibtramine, orlistat, mazindol, Rhubarb, Senna Leaf, etc. have been found as illegal constituents. In the tonic type products, we have identified more than 20 synthetic compounds relating to the erectile dysfunction (ED) treatment drugs, SIL, vardenafil and tadalafil (TDF). Since 2005, their synthetic intermediates and the patented but non-approved PDE5 inhibitors also have been found. It should be noted that TDF was found in the shells of capsule in 2009 and that mutaprodenafil was found as pro-drug type illegal component in 2010. In this report identification method of these illegal constituents is briefly described and then analytical trend in this decade is reviewed.

Rapid screening of illicit additives in weight loss dietary supplements with desorption corona beam ionisation (DCBI) mass spectrometry.

Desorption corona beam ionisation (DCBI), the relatively novel ambient mass spectrometry (MS) technique, was utilised to screen for illicit additives in weight-loss food. The five usually abused chemicals - fenfluramine, N-di-desmethyl sibutramine, N-mono-desmethyl sibutramine, sibutramine and phenolphthalein - were detected with the proposed DCBI-MS method. Fast single-sample and high-throughput analysis was demonstrated. Semi-quantification was accomplished based on peak areas in the ion chromatograms. Four illicit additives were identified and semi-quantified in commercial samples. As there was no tedious sample pre-treatment compared with conventional HPLC methods, high-throughput analysis was achieved with DCBI. The results proved that DCBI-MS is a powerful tool for the rapid screening of illicit additives in weight-loss dietary supplements.

Simultaneous analysis of psychotropic phenylalkylamines in oral fluid by GC-MS with automated SPE and its application to legal cases.

Phenylalkylamine derivatives, such as methamphetamine (MA), amphetamine (AM), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), phentermine (PT), fenfluramine (FFA) and phenmetrazine (PM), and ketamine (KT) are widely abused recreational or anorectic drugs in Korea and are regulated under the Controlled Substance Act in Korea. Phenylalkylamines and ketamine analysis is normally performed using both urine and hair samples but there is no established method for the simultaneous analysis of all these phenylalkylamines and ketamine in oral fluids. Oral fluid is easy to collect/handle and can provide an indication of recent drug abuse. In this study, to confirm the presence of phenylalkylamine derivatives and ketamine in oral fluid after screening with an immunoassay, an analytical method using automated solid phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS) was developed and fully validated according to international guidelines. The applicability of the assay was demonstrated by analyzing of authentic oral fluid samples and the results of oral fluid analysis were compared with those in urine and hair to to evaluate the feasibility of oral fluid in forensic cases. The recovery of phenylalkylamines and ketamine from oral fluid collection devices was also assessed. Oral fluid specimens from 23 drug abuse suspects submitted by the police were collected using Salivette (Sarstedt, Nümbrecht, Germany), Quantisal (Immunalysis, Pomona, CA) or direct expectoration. The samples were screened using a biochip array analyzer (Evidence Investigator, Randox, Antrim, UK). For confirmation, the samples were analyzed by GC-MS in selected-ion monitoring (SIM) mode after extraction using automated SPE (RapidTrace, Zymark, MA, USA) with a mixed-mode cation exchange cartridge (CLEAN SCREEN, 130 mg/3 ml, UCT, PA, USA) and derivatization with trifluoroacetic anhydride (TFA). The results from the immunoassay were consistent with those from GC-MS. Twenty oral fluid samples gave positive results for MA, AM, PT and/or PM among the 23 cases, which gave positive results in urine and/or hair. Although large variations in the MA, AM, PT and PM concentrations were observed in three different specimens, the oral fluid specimen was useful for demonstrating phenylalkylamines and ketamine abuse as an alternative specimen for urine.

Study on detection of drugs in slimming health foods using GC-MS/MS.

The determination of five drugs, fenfluramine (FEN), N-nitrosofenfluramine (NFE), sibutramine (SIB), mazindol (MAZ) and phenolphthalein (PHP), was studied in slimming health foods using GC-MS/MS. These drugs have been detected at high rates, especially in slimming health foods. Prolonged or excessive consumption of non-approved or unauthorized pharmaceuticals may cause serious adverse health consequences. In this study, samples were extracted with methanol and ultrasonication. Analyses were performed by GC-MS/MS, using established MS/MS parameters in the electron ionization (EI) mode and chemical ionization (CI) mode. In the EI mode, the recoveries of five drugs from several types of slimming health foods such as tablets, capsules and tea-bags spiked at 1 µg/mg (except PHP, spiked at 4 µg/mg) were in the range of 85.0-110.7% and 100 µg/mg (except PHP, spiked at 200 µg/mg) were 94.9-102.9%, respectively. In the CI mode, good recoveries of 80.3-102.2% (spiked at low concentration) and 92.8-103.2% (spiked at high concentration) were also obtained. We evaluated the present method using four slimming health foods, in which drugs had previously been detected. The results were similar to the previous results. These findings indicate that the present procedure for evaluating five drugs in slimming health foods by means of GC-MS/MS is useful.

Development of a liquid chromatography tandem mass spectrometry method for simultaneous determination of eight adulterants in slimming functional foods.

A method for simultaneous determination of eight adulterants including two appetite suppressants, two energy expenditure-enhancing drugs, one diuretic and three cathartics in slimming functional foods by high performance liquid chromatography with electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) was established. After samples were ultrasonically extracted with 70% (v/v) methanol aqueous solution and centrifuged, the components of ephedrine, norpseudoephedrine, fenfluramine, sibutramine, clopamide, emodin, rhein, and chrysophanol in sample solution were separated by a Hypersil Gold column (2.1 mm × 150 mm, 5 µm) using a programmed gradient elution. A mobile phase consisting of 0.02% (v/v) formic acid-ammonium formate buffer solution (pH=3.50) and methanol was used for elution with a flow rate set at 250 µL/min and column temperature of 25 °C. Qualitative determination was based on characteristic ion pairs and retention time of the targeted compounds using SRM (selective reaction monitoring) mode. Clenbuterol and ibuprofen were internal standards in positive and negative ionization mode, respectively. The internal standard curves were used for quantification measurement. The average recoveries of three different concentrations were from 80.2% to 94.5%. The limits of detection (LODs) were from 0.03 to 0.66 mg/kg (except chrysophanol 1.6 mg/kg). The linear dynamic range covered from 1 to 500 µg/L (except chrysophanol 50-5000 µg/L) for the twelve samples analyzed. Adulterants in four different kinds of slimming functional foods were determined by this developed method, and satisfactory results were obtained. These experimental results showed that, adulteration of sibutramine or/and fenfluramine were the major adulterating components with contents varying from 6.1 to 1.3×10(3) mg/kg and 1.9 to 9.7×10(3) mg/kg, respectively. In addition, three cathartic compounds were detected in six of those tested samples, and ephedrine, norpseudoephedrine and clopamide were not detected in all samples.

Quantification of fenfluramine with a molecularly imprinted chemiluminescence sensor and sulfonophenylazo rhodanine.

Fenfluramine-imprinted polymer was prepared by self assembly with acrylic amide as functional monomer and ethylene glycol dimethacrylate as crosslinker. The binding characteristics of the imprinted polymer to fenfluramine were evaluated by equilibrium binding experiments and the morphology was studied by SEM. Taking the imprinted polymer as recognition material, using the new 3-p-nitrylphenyl-5-(4'-methyl-2'-sulfonophenylazo)rhodanine (M4NRASP) synthesized by the authors as chemiluminescence reagent, a new highly selective flow injection chemiluminescence sensor for trace fenfluramine with good sensitivity was established which utilized the chemiluminescence reaction between fenfluramine, M4NRASP, and potassium permanganate in hydrochloric acid. The traditional flow-through cell was replaced with a polymethyl methacrylate module, with Y-shaped flow path, through which the above three reactants were injected simultaneously. Under optimum conditions the relative chemiluminescence intensity shows a linear relationship with the concentration of fenfluramine over the range of 5 x 10(-7) to 8 x 10(-6) g/mL with a lower detection limit of 3.4 x 10(-8) g/mL. The relative standard deviation for the determination of 1.0 x 10(-6) g/mL fenfluramine solution was 2.4% (n = 11). This proposed sensor could be satisfactorily applied to the determination of fenfluramine in weight-reducing capsules.

Determination of fenfluramine based on potassium permanganate-calcein chemiluminescence system.

Calcein was found to be able to use as chemiluminescence reagent and post-chemiluminescence was observed when fenfluramine was injected into the mixture after the CL reaction of calcein-potassium permanganate. Based on this phenomenon, a flow injection CL method was established for the determination of fenfluramine. The possible CL mechanism was proposed. The CL intensity was correlated linearly with the concentration of fenfluramine over the range of 1.0 x 10(-7) to 6.0 x 10(-6) g mL(-1) and the detection limit was 6 x 10(-8) g mL(-1). The relative standard deviation was 2.2% for 5.0 x 10(-7) g mL(-1) fenfluramine (n=11). This method was applied to the determination of fenfluramine in weight-reducing tonic successfully.

Adulteration of over-the-counter slimming products with pharmaceutical analogue--an emerging threat.

OBJECTIVES: To review pharmaceutical analogue adulteration of non-prescription slimming products. DESIGN: Retrospective study. SETTING: Tertiary referral centre for toxicology analysis, Hong Kong. PATIENTS: All patients known to have been hospitalised after taking slimming products adulterated with pharmaceutical analogues from September 2004 to December 2006. MAIN OUTCOME MEASURES: Age, reasons for hospital admission, major biochemical findings, and toxicology analysis results of the offending slimming products. RESULTS: N-nitrosofenfluramine, an analogue of fenfluramine with hepatotoxic effect, was found in two slimming products. Three patients were hospitalised after taking these slimming products, one of whom developed liver failure treated by liver transplantation. Another slimming product was found to contain N-desmethyl-sibutramine, an analogue of sibutramine. A patient with an unremarkable medical history developed acute psychosis after taking the latter product for 1 week. CONCLUSIONS: Analogues, created by modifying the chemical structures of pharmaceuticals, are used as adulterants in non-prescription slimming products, in an attempt to evade regulatory inspection. The imperceptible use of these analogues is very dangerous because they have not been tested formally for efficacy and safety. In view of the potential harm to the public, more effective and proactive measures are required to guard against the illicit use of pharmaceutical analogues. There is also a need for increased awareness among the public and the medical professionals about this emerging threat.

Quantitative 1H NMR method for the routine spectroscopic determination of enantiomeric purity of active pharmaceutical ingredients fenfluramine, sertraline, and paroxetine.

Determining the enantiomeric purity of chiral therapeutic agents is important in the development of active pharmaceutical ingredients (API). A strategy for determining the enantiomeric purity of three APIs was developed using nuclear magnetic resonance (NMR) and the chiral solvating agent (CSA) 1,1-bi-2-naphthyl (1). While chiral chromatography is widely used to evaluate enantiomeric purity, it can sometimes suffer from tedious sample preparation obviating rapid measurements that are sometimes needed during the manufacture of such agents. The techniques described herein provide comparable enantiomeric purity results with those obtained with traditional chiral HPLC and other published methods for these compounds. Chiral analysis of standard samples of methylbenzylamine enantiomeric mixtures using 1 were found to be quantitative to approximately 1% minor enantiomer. Enantiomeric purity determination by NMR utilizing chiral solvating agents do not require special instrumental techniques, chemical derivatization or standards and is therefore ideally suited for rapid routine analysis. As a result, the technique demonstrated is commonly used in our laboratory as a complementary or alternative method to chiral HPLC or optical rotation measurements for routine determination of enantiomeric purity.

Identifying N-nitrosofenfluramine in a nutrition supplement.

The analytical data for identifying an unknown substance that was found in a nutrition supplement is presented. The unknown substance is purified using thin-layer chromatography and then measured using high-resolution mass spectrometry (HRMS) giving the exact mass from which the structure of the unknown substance was proposed. The procedure for synthesizing N-nitrosofenfluramine from fenfluramine is described. The extracted, synthesized, and standard N-nitrosofenfluramine are compared using HRMS, high-performance liquid chromatography (HPLC)-MS, HPLC-UV, Fourier transform IR spectroscopy, gas chromatography-MS, TLC, and NMR (1H NMR and 13C NMR). All analytical data obtained confirm that the unknown peak in the nutrition supplement is N-nitrosofenfluramine and that the synthetic procedure described can easily provide the N-nitrosofenfluramine reference substance for identification.

Hair analysis for fenfluramine and norfenfluramine as biomarkers for N-nitrosofenfluramine ingestion.

In this paper, a high performance liquid chromatographic method with fluorescence detection (HPLC-FL) for the determination of fenfluramine (Fen) and norfenfluramine (Norf) in human hair as biomarker metabolites of N-nitrosofenfluramine (N-Fen) is described. Washed and cut hair segments were extracted by ultrasonication for 1h at room temperature in methanol. The extract was evaporated and applied for derivatization with the fluorescent reagent 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl). An HPLC-FL analysis was performed using an ODS column with mobile phase composition of acetonitrile and water (65:35, v/v) and monitored at 430 nm (excitation 325 nm). The method was sensitive with detection limits of 36 and 16 pg/mg hair for Fen and Norf, respectively. The linearity was assessed in the range 0.036-144 ng/mg for Fen and 0.016-127 ng/mg for Norf with correlation coefficients larger than 0.999. The method was successfully used for the segmental determination of Fen and Norf in hair samples obtained from hospitalized patients diagnosed with hepatotoxicity and suspected to ingest N-Fen. Both Fen and Norf could be detected in these patients' hair samples in the ranges 43-1389 pg/mg for Fen and 18-680 pg/mg for Norf and the results showed that the patients might ingest N-Fen for a period of not less than 5 months. As well, the method was applied for the determination of Fen and Norf in rats that possess pigmented and non-pigmented hair after an intraperitoneal administration of Fen. Both compounds were determined in black as well as in white hair.

A fatal case of hepatic failure possibly induced by nitrosofenfluramine: a case report.

A 42-year-old female developed fulminant hepatic failure after having ingested an undetermined quantity of a herbal product over a period of approximately four months prior to the onset of her illness. Clinically, the cause of liver failure was assessed to be drug-induced and she eventually underwent total hepatectomy, with porto-caval shunting, in anticipation of a living-unrelated liver transplant. Unfortunately, her condition deteriorated and she died less than 48 hours post-operatively, approximately three weeks post-admission. An autopsy showed that the subject was deeply jaundiced and severely obese (BMI: 47.1 kg m(-2)), with evidence of diffuse haemorrhage, including the presence of 1.35 l of blood in the peritoneal cavity. The liver had been removed and was later recovered as a formalin-fixed specimen which was markedly contracted, comprising multiple micronodules interspersed with extensive areas of dense fibrotic tissue. Histologically, there was massive necrosis of the hepatic parenchyma, such that the residual hepatocytes were disposed as nodules displaying variable cellular regeneration and ballooning degeneration, attended by florid ductal proliferation and mixed inflammatory infiltrates. Infective, autoimmune, metabolic, vascular, neoplastic and most other natural causes of massive hepatocellular necrosis were effectively excluded. Analysis of the post-mortem blood samples yielded fluconazole, metronidazole, frusemide, lignocaine and tramadol, (therapeutic agents administered to the patient during her last illness). Subsequent analysis of the residual capsules revealed that they were adulterated by fenfluramine, N-nitrosofenfluramine (1.3-1.6 mg per capsule), nicotinamide (13.3-15.6 mg per capsule) and thyroid extract. None of the herbal ingredients is currently known to be hepatotoxic and much the same applies to fenfluramine, nicotinamide (except when taken in mega-doses) and thyroid extract. However, as nitrosamines are known to be variably hepatotoxic, it would be reasonable to surmise that, in the absence of a more plausible cause of liver damage, N-nitrosofenfluramine was the likely cause of massive hepatocellular necrosis in this instance.

[Determination of fenfluramine, diethylpropion and mazindol in slimming foods by gas chromatography-mass spectrometry].

A gas chromatography-mass spectrometry (GC/MS) analytical method for three anorectics drugs, fenfluramine, diethylpropion and mazindol, illegally adulterated in slimming foods has been developed. They can be simultaneously identified and quantified. The samples were extracted with chloroform, and then separated by GC with an HP-5MS GC capillary column. The temperature programming was started at 70 degrees C. The temperature was held at this temperature for 2 min and then increased at 10 degrees C/min to 280 degrees C. The completely separated peaks were identified by MS with a quadrupole mass filter and quantitated in selected ion monitoring mode. Compared with the NIST98 library, the matching qualities of the drugs in foods were all over 90%. The calibration curves of the drugs were excellent with correlation coefficients between 0.9991 and 0.9999, and relative standard deviations between 1.6% and 2.2%. The recoveries were between 96.0% and 102.4%. The results demonstrated that this method is suitable for the identification and quantitation of these anorectics drugs in slimming foods.

[Determination of the optical purity of N-nitrosofenfluramine found in the Chinese slimming diet].

From 2001 to the summer of 2002, more than 800 cases of liver damage were reported in Japan among people taking Chinese diet aids. The Japanese Ministry of Health, Labor and Welfare has recently announced that N-nitrosofenfluramine was the hepatotoxic compound contained in the diet aids based on animal experiments performed by the National Institute of Health Sciences. Although N-nitrosofenfluramine is a derivative of fenfluramine, a previously used antiobesity drug, neither pharmacologic nor toxicologic properties have been reported for N-nitroso fenfluramine. It should be noted that N-nitrosofenfluramine has two optical isomers, although it is not yet known which isomer damages the liver and other organs. The Japanese Ministry of Health, Labor and Welfare has not commented on this point. Pursuing this question, 10 types of Chinese slimming aid samples including those obtained from patients with fulminating hepatitis were analyzed by NMR, GC/MS, and a newly established HPLC method using a chiral separation column. It was found that the N-nitrosofenfluramine in all of the toxic diet aids was the (S)-isomer form. No (R)-isomer was detected. These results strongly suggest that the nitroso-compound in the diets must be prepared from pharmacologically active (S)-fenfluramine (dexfenfluramine). Thus the pharmacologic and toxicologic properties of each isomer should be investigated.

[Enantioseparation of twelve pharmaceutical racemates with high performance capillary electrophoresis using L-leucine as chiral selector].

A rapid enantiomeric separation method using L-leucine as chiral selector was established. Capillary zone electrophoresis (CZE) has been used for the enantiomeric separation of twelve pharmaceutical racemates with bare fused silica capillary and employing L-leucine as chiral selector. The enantiomeric resolution was influenced by L-leucine concentration and pH of background electrolyte (BGE). The effects of the BGE types and concentrations on the enantiomeric separation were also investigated. The results showed that in the solution containing 50 mmol/L borax and 70 mmol/L L-leucine (pH 9.0), all the twelve drugs were on baseline separated in less than 11 minutes.

[Determination of fenfluramine hydrochloride in tablets by gas chromatography with derivatization].

The contents of fenfluramine hydrochloride in tablets were determined by using gas chromatography (GC) with derivatization. Five percent SE-30 was used as stationary liquid(2.1 m x 3.2 mm i.d., glass column), and was detected with flame ionization detector (FID). The oven temperature was set at 125 degrees C, with injector and FID temperature at 160 degrees C. The fenfluramine hydrochloride in the tablets was extracted with ethyl acetate and derivatized with trifluoroacetic anhydride at 40 degrees C for 30 min, and then analyzed by GC. The quantitative determination was performed with mexiletine as the internal standard. The method showed good linearity within the mass concentration range from 0.1 g/L-0.5 g/L(r = 0.9996) and the limit of detection was 8 ng. The average recovery of the samples was (100.2 +/- 2.2)% (n = 6). The precision of the method is satisfactory(RSD = 1.4%). The method developed has been applied to the determination of fenfluramine hydrochloride in tablets.

[Determination of fenfluramine in corpse using GC/NPD and GC/MS].

A method was described for the determination of fenfluramine by GC/MS in blood, urine, liver, lung, kidney and heart tissues. After liquid-liquid extraction of fenfluramine in the specimens obtained from autopsy, the samples were qualitatively identified by GC/MS, and quantitatively determined by GC/NPD. 4-phenyl butylamine was used as internal standard. The concentrations of fenfluramine in blood, urine and liver were 7.8 micrograms/ml, 64.2 micrograms/ml and 31.3 micrograms/g respectively. The findings of autopsy and the results of toxicological analysis were also discussed.