Screening for forensically relevant benzodiazepines in human hair by gas chromatography-negative ion chemical ionization-mass spectrometry

A procedure is presented for the detection in human hair of forensically relevant benzodiazepines, i.e. nordiazepam, oxazepem, bromazepam, diazepam, lorazepam, flunitrazepam, alprazolam and triazolam. The method involves decontamination of hair with methylene chloride, pulverization in a ball mill, incubation of 50 mg powdered hair in Soerensen buffer (pH 7.6) in the presence of prazepam-d₅ used as internal standard, liquid-liquid extraction with diethyl ether-chloroform (80:20, v/v) and gas chromatography-mass spectrometry using negative chemical ionization after derivatization with, N,O-bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane. The limits of detection for all benzodiazepines ranged from 1 to 20 pg/mg using a 50-mg hair sample. Coefficients of variation and extraction recoveries, ranging from 7.4 to 25.4% and 47.6 to 90%, respectively, were found suitable for a screening procedure. One hundred and fifteen samples were submitted to this screening procedure, and specimens tested positive for nordiazepam (0.20-18.87 ng/mg, n = 42) and its major metabolite oxazepam (0.10-0.50 ng/mg, n = 14), flunitrazepam (19–148 pg/mg, n = 31), lorazepam (31–49 pg/mg, n = 4) and alprazolam (0.3-1.24 ng/mg, n = 2). Bromazepam, diazepam and triazolam were not detected.     

Development of a reference material using methamphetamine abusers' hair samples for the determination of methamphetamine and amphetamine in hair

In the present study, we developed a reference material (RM) using authentic hair samples for the determination of methamphetamine (MA) and its main metabolite, amphetamine (AP) in human hair. MA abusers' hair samples were collected, homogenized and finally bottled. The concentration of each bottle was determined using two extraction methods, agitation with 1% HCl in methanol at 38 degrees C and ultrasonication with methanol/5M HCl (20:1), followed by gas chromatography/mass spectrometry (GC-MS) after derivatization with trifluoroacetic anhydride (TFAA). Both analytical procedures were fully validated and their extraction efficiency was compared. The homogeneity of analytes was evaluated and their property values were determined with their uncertainties. The two methods were acceptable to analyze MA and AP in human hair through the validation and comparative studies using spiked and authentic hair samples as well as NIST SRM 2379 certified reference material. Satisfying homogeneity was reached for MA and AP in the prepared RM. Finally, a human hair RM containing MA and AP is prepared at the level of 7.64+/-1.24 and 0.54+/-0.07 ng/mg, respectively. This material can be useful in forensic laboratories for internal quality control and external quality assurance.     

Application of ion mobility spectrometry to the rapid screening of methamphetamine incorporated in hair

Using ion mobility spectrometry (IMS), a simple, sensitive and rapid screening for methamphetamine (MA) incorporated in user's hair has been developed. To completely unbind MA from hair matrix and to achieve its effective vaporization for the IMS detection, the hair sample was digested in 5 M NaOH (methanol-water, 4:1, v/v) solution prior to IMS measurement. MA in hair was semi-quantitatively detected by monitoring the digested hair sample employing dibenzylamine (DBA) as internal standard. The minimum amount of hair sample required was 2 mg and its digested sample was ample for four IMS measurements. Teh detection limit of MA in hair was 0.5 ng mg⁻¹. This proposed method was applicable to the semi-quantitative detection of MA in users' hair samples, and to the sectional analysis for MA in a limited amount of user's hair. The IMS results obtained were in good agreement with their GC-MS determination.     

Testing of the anabolic stanozolol in human hair by gas chromatography–negative ion chemical ionization mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of stanozolol in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride and the sonication in methanol of 100 mg of powdered hair for 2 h. After elimination of the solvent, the hair sample was solubilized in 1 ml 1 M NaOH, 15 min at 95°C, in the presence of 10 ng stanozolol-d₃ used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C₁₈) and a liquid–liquid (pentane) extraction. After evaporation of the final organic phase, the dry extract was derivatized using 40 μl MBHFA–TMSI (1000:20, v/v), incubated for 5 min at 80°C, followed by 10 μl of MBHFBA, incubated for 30 min at 80°C. The derivatized extract was analyzed by a Hewlett-Packard GC–MS system with a 5989 B Engine operating in the negative chemical ionization mode of detection. Linearity of the detector response was observed for stanozolol concentrations ranging from 5 to 200 pg/mg with a correlation coefficient of 0.998. The assay was capable of detecting 2 pg of stanozolol per mg of hair when approximately 100 mg hair material was processed, with a quantification limit set at 5 pg/mg. Intra-day precision was 5.9% at 50 pg/mg and 7.8% at 25 pg/mg with extraction recoveries of 79.8 and 75.1%, respectively. The analysis of a 3-cm long hair strand, obtained from a young bodybuilder (27 year old) assuming to be a regular user of Winstrol (stanozolol, 2 mg), revealed the presence of stanozolol at the concentration of 15 pg/mg.     

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.     

Simultaneous enantioselective determination of amphetamine and congeners in hair specimens by negative chemical ionization gas chromatography-mass spectrometry

Enantioselective quantification of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) enantiomers in hair using gas chromatography-mass spectrometry (GC-MS) is described. Hair specimens were digested with 1M sodium hydroxide at 100 degrees C for 30 min and extracted by a solid phase procedure using Cleanscreen ZSDAU020. Extracted analytes were derivatised with (S)-heptafluorobutyrylprolyl chloride and the resulting diastereoisomers were quantified by GC-MS operating in the negative chemical ionization mode. Extraction yields were between 73.0 and 97.9%. Limits of detection varied in the range of 2.1-45.9 pg/mg hair, whereas the lowest limits of quantification varied between 4.3 and 91.8 pg/mg hair. Intra- and inter-assay precision and respective accuracy were acceptable. The enantiomeric ratios (R versus S) of AM, MA, MDA, MDMA and MDEA were determined in hair from suspected amphetamine abusers. Only MA and AM enantiomers were detectable in this collective and the quantification data showed in most cases higher concentrations of (R)-MA and (R)-AM than those of the corresponding (S)-enantiomers.     

Routine analysis of amphetamine and methamphetamine in biological materials by gas chromatography-mass spectrometry and on-column derivatization

A simple determination method of amphetamine (AP) and methamphetamine (MA) in biological materials was developed using on-column derivatization and gas chromatography-mass spectrometry (GC-MS). AP and MA in biological materials were adsorbed on the surface of Extrelut and then extracted and derivatized simultaneously on the Extrelut column. AP and MA were derivatized to the N-propoxycarbonyl derivatives using propylchloroformate. Pentadeuterated MA was used as an internal standard. The recoveries of AP and MA from urine were 88.2 and 92.5%, and those from blood were 89.7 and 90.3%, respectively. The calibration curves showed linearity in the range of 12.5-2000 ng/ml (ng/g) for AP and MA in urine and blood, and 0.25-20 ng/mg in hair. When urine samples containing two different concentrations (200 and 1000 ng/ml) of AP and MA, blood samples containing two different concentrations (200 and 1000 ng/g) of AP and MA, hair samples containing two different concentrations (0.5 and 5.0 ng/mg) of AP and MA, the coefficients of variation of intra-day and inter-day were 0.68-3.60% in urine, 0.42-4.58% in blood, and 1.20-13.1% in hair. Furthermore, this proposed method was applied to a medico-legal case of MA intoxication.     

Single hair analysis of methamphetamine and amphetamine by solid phase microextraction coupled with in matrix derivatization

A sensitive method for detection of methamphetamine (MA) and amphetamine (AP) in human hair was developed using solid phase microextraction (SPME) and one-pot derivatization. MA and AP were directly derivatized to N-propoxycarbonyl derivatives in an aqueous solution by propylchloroformate in a one-pot reaction before extraction by SPME. The derivatives were extracted to a coating of SPME from a headspace of the vial. The adsorbed derivatives were thermally desorbed in the injection port of a gas chromatograph. Pentadeuterated MA was used as an internal standard. The absolute recoveries of MA and AP from the spiked hair were 2.80-17.5%, respectively. The calibration curves showed linearity in the range of 0.05-20 ng/0.08 mg/vial for MA and 0.1-20 ng/0.08 mg/vial for AP in hair. Detection limits (S/N = 3) of MA and AP were 0.02 and 0.05 ng/0.08 mg/vial. The coefficients of variation of intraday were 1.04-26.4%. Additionally, this proposed method was applied to segmental analysis in clinical and medico-legal cases of MA intoxication.     

Gas chromatographic analysis of nicotine and cotinine in hair

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography—mass spectrometry. Hair (about 50–100 mg) was incubated in 1 M sodium hydroxide at 100°C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Simultaneous determination of amphetamine,methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry

A procedure is presented for the simultaneous identification and quantification of amphetamine (AP), methamphetamine (MA), methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) in human hair. The method involves decontamination of hair with dichloromethane and warm water, heat-alkaline hydrolysis in the presence of deuterated internal standards, liquid-liquid extraction and gas chromatography-mass spectrometry after derivatization with pentafluoropropionic anhydride-pentafluoropropanol. The limit of detection for AP, MA and MDA was 0.05 ng/mg using a 50-mg hair sample; for MDMA it was 0.1 ng/mg. Coefficients of variation ranged from 7 to 18%. This assay has been successfully utilized in the evaluation of the deposition of the drugs in hair obtained from various parts of the anatomy of a stimulant abuser.     

The dependence of the incorporation of methamphetamine into rat hair on dose,frequency of administration and hair pigmentation

In this paper, the incorporation of methamphetamine (MA) into rat hair was studied. The main purpose of this study was to investigate whether MA can be detected or positive hair results can be obtained in hair of rats administered a single dose of MA. The relationship between dose and frequency of administration and the concentrations of MA and its metabolite, amphetamine (AP), in rat hair were evaluated and the MA and AP concentrations in white and pigmented hair were compared. MA was administered to rats as follows: low dose (0.5 mg/kg/day), medium dose (2 mg/kg/day) and high dose (10 mg/kg/day). The frequency of administration was one time per day for 1, 2, 3, 4, 5, 15 and 30 days. Hair and urine samples were collected from rats and analyzed by gas chromatography/mass spectrometry (GC/MS). MA could be identified in pigmented rat hair when MA was administered for 4 or more days at low daily dose and on day 1 following administration of medium and high daily doses. Positive results for MA were obtained from pigmented rat hair when MA was administered for 30 days at low daily dose, for 4 or more days at medium daily dose, or for 2 or more days at high daily dose. The concentrations of MA and AP found in rat hair were proportional to the dose and frequency of administration. The concentrations of MA and AP in pigmented rat hair were 2–10 times higher than those in white rat hair. The results of this study on the incorporation of MA into rat hair can serve as a model to better understand the incorporation of MA into human hair even though there are differences between animal models and human hair.     

Supercritical fluid extraction of drugs in drug addict hair

Opiates were extracted from sixteen hair samples of drug addicts using a supercritical fluid extraction method with supercritical carbon dioxide and a modifier solution of methanol-triethylamine-water (2:2:1, v/v). The concentrations, as determined by GC-MS, ranged from 1.22 to 21.73 (mean 7.60 ng/mg), 0.17 to 1.54 (mean 0.69 ng/mg) and 0.15 to 14.09 ng/mg hair (mean 3.78 ng/mg) for codeine, morphine and 6-monoacetylmorphine, respectively. The reproducibility of the total procedure had a relative standard deviation of 13%, 17% and 14% for codeine, morphine and 6-monoacetylmorphine, respectively. By this method, concentrations of 0.3, 0.2 and 0.1 ng/mg hair for codeine, morphine and 6-monoacetylmorphine, respectively, could be detected. Relative extraction recoveries were 61%, 53% and 96% for codeine, morphine and 6-monoacetylmorphine, respectively.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

A preliminary study of steroid reproductive hormones in human hair

Nowadays research and clinical studies of human reproductive endocrinology are generally carried out using human blood reproductive hormone assays. However the acquisition of human blood samples has some shortcomings. In search of new approaches, we paid attention to the fact that progesterone can be detected in cow's hair. Consequently we investigated whether or not steroid hormones are measurable in human hair. The results showed that the levels of steroid hormones in hair are not affected by shampoo and do not significantly vary between different segments of hair (i.e. top, middle and basal segments). The menstrual estradiol and progesterone rhythm of female hair is similar to that of female serum. The ratio of hair estradiol to serum estradiol in the female is 41.2% and that of hair progesterone to serum progesterone is 59.0%; the ratio of hair testosterone to serum testosterone in male is 116%. There are significant correlations between hair and serum steroid hormones of healthy human adult: γ (estradiol)=0.395 (n=20), p<0.05; γ (progesterone)=0.440 (n=22), p<0.025 and γ (testosterone)=0.395 (n=25), p<0.05.     

Clara cell protein (CC16) in serum and bronchoalveolar lavage fluid of subjects exposed to asbestos

The Clara cell protein (CC16) is a small and readily diffusible protein of 16kDa secreted by bronchiolar Clara cells in the distal airspaces. These epithelial cells are altered in several pulmonary pathological processes induced by various lung toxicants. In the search for a new biomarker of asbestos-induced lung impairment, we used a sensitive immunoassay to determine the levels of CC16 in bronchoalveolar fluid (BALF) and serum of subjects exposed to asbestos compared with a group of healthy controls. In the BALF of asbestos-exposed subjects there was an insignificant trend towards CC16 elevation compared with controls, with a (mean ±SD of 0.81 ±0.65mg l^-1 for asbestos-exposed subjects (n = 23) versus 0.39 ±0.19mg l^-1 for controls (n = 11) (p = 0.09). In serum, CC16 concentration was significantly increased among asbestos-exposed subjects, with values of 27.2 ±24.0 µg l^-1 for asbestos-exposed subjects (n = 34) versus 16.1 ±7.6 µg l^-1 for controls (n = 34) (p = 0.01). Regarding the effects of smoking, there were significant differences between generally lower CC16 levels in serum and BALF (p = 0.05 and 0.001, respectively) of smokers compared with the higher levels in non-smokers. Serum CC16 levels positively correlated with those in BALF, which is consistent with a diffusional transfer of CC16 from the bronchoalveolar space into the serum. No association, however, emerged between the levels of CC16 in serum or BALF and either the duration of asbestos exposure or the severity of the lung impairment as assessed by chest X-ray. These findings suggest that exposure to asbestos elicits early changes in the local and, importantly, also the systemic levels of CC16. This pneumoprotein therefore appears as a promising non-invasive biomarker of asbestos-induced lung injury and occupational disease in both smoking and non-smoking exposed subjects.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Lymphocyte HSP72 following exercise in hyperthermic runners: The effect of temperature

Heat shock protein 72 (HSP72) is the most inducible HSP, but is not always increased in lymphocytes following exercise. This field study examined whether lymphocyte HSP72 was increased in hyperthermic (T_rec>39.0 °C) male athletes following a 14 km competitive race in cool conditions (ambient temperature 11.2 °C). A comparison was also made between control runners (n=7) and those treated for exertional heat illness (n=9). Lymphocyte HSP72 was not increased in control runners immediately post- compared with pre-race, and there was no difference between both groups of runners. A second study of the race (ambient temperature 14.6 °C) found that lymphocyte HSP72 in control (n=7) and treated (n=9) athletes was higher 2 days post- compared with immediately post-race (p<0.01) and these increases were correlated with post-exercise T_rec (p<0.05).     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

High-performance liquid chromatographic determination of bupivacaine in plasma samples for biopharmaceutical studies and application to seven other local anaesthetics

A sensitive analytical procedure for bupivacaine dosing in plasma samples by reversed-phase high-performance liquid chromatography is described. After a two-step extraction, the analysis was performed using a C₁₈ column and a mobile phase of 0.01 M sodium dihydrogen-phosphate (pH 2.1)—acetonitrile (80:20, v/v). The extraction yield of bupivacaine from plasma was 73.5 ± 5.1% (mean ± S.D., n = 10). The within-day and between-day reproducibilities at a concentration of 100 ng/ml were 2.1% and 5.6%, respectively (n = 10). Calibration curves were linear (r² = 0.9996) between 5 and 1000 ng/ml. The limit of detection, defined by a signal-to-noise ratio of 3:1, was 2 ng/ml. The accuracy at a concentration of 100 ng/ml was 2.3%. This method could be applied to the plasma analysis of seven other local anaesthetics (articaine, etidocaine, lidocaine, mepivacaine, pramocaine, procaine and tetracaine). The procedure was used in bioavailability studies of bupivacaine-loaded poly( -lactide) (i.e. PLA) and poly( -lactide-co-glycolide) (i.e. PLGA) microspheres after subcutaneous and intrathecal administrations in rabbits.