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A method for simultaneous bioanalysis of the three cytotoxic drugs cytosine arabinoside, daunorubicin and etoposide in human plasma was developed and validated. A HPLC method with ultra-violet and fluorescence detection, preceded by mixed-mode cation-exchange solid phase extraction sample preparation, was used for the quantification of the analytes. The assay was used for the simultaneous measurement of cytosine arabinoside, daunorubicin and etoposide with linearity in the ranges of 13–1500 ng/mL, 15–1000 ng/mL and 52.5–3500 ng/mL, respectively. The chromatographic run-time was 15.5 min. The overall precision (% relative standard deviation) was within 0.2–13.5% and the recovery ranged between 86.1% and 110.1% for the three drugs at all concentrations tested. Plasma samples were stable for at least two months when stored at −20 °C. The method was successfully applied to quantification of the three drugs in blood samples from patients undergoing induction treatment for acute myeloid leukaemia, thus demonstrating its suitability for clinical studies.

Many patients with psychotic symptoms respond poorly to treatment. Factors possibly affecting treatment response include the presence of polymorphisms in genes coding for various receptor populations, drug-metabolizing enzymes or transport proteins.To investigate whether genetic polymorphisms could be indicators of treatment response to antipsychotic drugs. The genes of interest were the dopamine D2 receptor gene (DRD2), the serotonin 2A and 2C receptor genes (HTR2A and HTR2C), the P-glycoprotein gene (ABCB1 or MDR1) and the drug-metabolizing cytochrome P450 2D6 gene (CYP2D6).Data for this naturalistic, cross-sectional study of patients requiring antipsychotic drugs and attending the Psychosis Outpatient Care clinic in Jönköping, Sweden were obtained from patient interviews, blood samples and information from patient files. Blood samples were genotyped for DRD2 Taq1 A, Ins/Del and Ser311Cys, HTR2A T102C, HTR2C Cys23Ser, ABCB1 1236CT, 2677GT/A, 3435CT and genetic variants of CYP2D6. The patients (n=116) were grouped according to the CANSEPT method regarding significant social and clinical needs and significant side effects.Patients on olanzapine homozygous for ABCB1 3435T, had more significant social and clinical needs than others. Patients with one or two DRD2 Taq1 A1 alleles had a greater risk of significant side effects, particularly if they were male, Caucasian, had a schizophrenic or delusional disorder or were taking strong dopamine D2-receptor antagonistic drugs.If these results are confirmed, patients carrying the DRD2 Taq1 A1 allele would benefit from using drugs without strong dopamine D2 receptor antagonistic properties.

Although numerous studies have dealt with changes in blood brain-derived neurotrophic factor (BDNF), methodological issues about BDNF measurements have only been incompletely resolved. We validated BDNF ELISA with respect to accuracy, reproducibility and the effect of storage and repeated freezing cycles on BDNF concentrations. Additionally, the effect of demographic characteristics in healthy subjects on BDNF was verified. Whole blood and serum was collected from 206 healthy subjects and a subgroup was genotyped for BDNF Val66Met polymorphism. The effect of age, gender, BDNF genotype and thrombocyte count on whole blood BDNF was assessed. The BDNF ELISA measurement was accurate, 91.6+/-3.0%, and showed high reproducibility, whereas inter-assay and intra-subject variations were modest, 8.4+/-5.2% and 17.5+/-14.1%, respectively. Storage of whole blood samples at 4 degrees C significantly decreased BDNF concentration, while repeated freezing cycles and storage at -20 degrees C was without any effect. Storage at -20 degrees C of serum, but not whole blood, was associated with a significant decrease in BDNF concentration. Women had significantly higher whole blood BDNF concentrations than men (18.6+/-1.3 ng/ml versus 16.5+/-1.4 ng/ml), and showed a right-skewed BDNF concentration distribution. No association between whole blood BDNF concentrations and thrombocyte count, age, or BDNF genotype was found. In conclusion, the BDNF ELISA assay determines whole blood BDNF accurately and with high reproducibility. Female gender is associated with higher whole blood BDNF concentrations whereas age, thrombocyte count and BDNF Val66Met polymorphism were un-associated.

The serotonin transporter radioligand [11C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, or [11C]DASB, was examined in order to assess its potential for measuring fluctuations in endogenous serotonin concentrations with positron emission tomography. Binding characteristics of [11C]DASB and the propensity for serotonin to displace the tracer were explored in rat brain homogenates. Experiments showed that serotonin displaced [11C]DASB in vitro. Ex vivo experiments performed after tranylcypromine injection (3 or 15 mg/kg) showed a dose-dependent trend in radioactivity uptake and suggested that serotonin may compete with [11C]DASB for transporter binding.

The non-invasive radiotracer technique positron emission tomography (PET) may provide valuable information in the toxicokinetic-toxicodynamic evaluation of endogenous or toxic environmental compounds. Assessment of mechanism of action of toxins is often difficult to validate. In this respect, PET may offer advantages since it can quantify not only the distribution and kinetics of the radiolabelled toxin in the body, but also the altered rates of physiological or biochemical processes induced by the toxin. It is even possible to validate the body distribution and tissue accumulation of the toxic compound in primates, since linear kinetics can be assumed after administration of the radiolabelled compound in minute amounts without any toxic or physiological effects. Quantitative estimates can be derived with accuracy and high precision. Using a multi-tracer protocol, it is often possible to illuminate both the kinetics and the dynamics of a toxic compound. Long-term effects of different toxins on dopamine receptor function have been evaluated with PET as well as the influence of Parkinson disease medication on pre- and postsynaptic dopaminergic receptor function over the course of the disease. In conclusion, PET may provide very informative insight into complex receptor interactions of both toxic compounds and drugs under development.

The substituted (S)-3-phenylpiperidine (−)-OSU6162 belongs to a novel class of functional modulators of dopaminergic systems. In vivo, (−)-OSU6162 has a unique stabilising profile on dopaminergic functions. In vitro this compound exhibits low affinity for the dopamine D2 receptor, but due to its similarity to neuroleptics on brain dopaminergic neurochemistry and different postsynaptic effects it has been characterised as a preferential dopamine autoreceptor antagonist. To further clarify the effects of (−)-OSU6162 on the postjunctional nigrostriatal dopaminergic system, dopamine receptor binding was measured in rhesus monkeys (Macaca mulatta) by positron emission tomography (PET) using the D1 and D2 dopamine receptor radioligands [11C]SCH23390 and [11C]raclopride respectively, before and during continuous intravenous infusions of (−)-OSU6162. Additionally, the test-retest variability of sequential [11C]SCH23390 scans was estimated. Following the administration of (−)-OSU6162, [11C]raclopride binding in striatum was dose-dependently decreased with a 76% reduction occurring after 3.0 mg/kg per h continuous infusion. Whereas (−)-OSU6162 in the lower doses had no effect on [11C]SCH23390 binding, the highest dose, 3.0 mg/kg per h, increased [11C]SCH23390 binding, which may indicate a potentiating effect on D1 dopamine receptor mediated functions. Thus, in contrast to the conditions in vitro, (−)-OSU6162 produces a high displacement of raclopride from D2 receptors in vivo.

The labelling of the presynaptic dopamine receptor antagonist (-)-OSU6162, ((S)-(-)-3-(3-(methylsulfonyl)phenyl)-1-propylpiperidine) was performed by an alkylation with [11C]methyl iodide of the thio anion (-)-OSU1281, followed by a selective oxidation to the corresponding methyl sulfone, [11C-methyl]-(-)-OSU6162. The total radiochemical yield calculated from the produced [11C]carbon dioxide to final product was about 25% and the time of synthesis was in the range of 40 min from end of bombardment. The synthesis of the precursor, (-)-OSU1281, was performed from (-)-3PPP in a three-step synthesis. The regional brain distribution of (-)-OSU6162 radiolabelled with 11C was studied in rhesus monkeys by means of positron emission tomography, PET. [11C-Methyl]-(-)-OSU6162 was rapidly and uniformly distributed to gray matters of the brain, and no decrease of radioactivity uptake in the brain was seen after pretreatment with 1 to 3 mg/kg/h of (-)-OSU6162. The effect of doses of 1 to 3 mg/kg/h of (-)-OSU6162 on the dopamine binding was studied by PET using [11C-methyl]raclopride. Radioactivity in the striatum was significantly and dose-dependently decreased by (-)-OSU6162 (r = 0.88), supporting competition with dopamine for selective binding to dopamine receptors.

The specific binding of N-methyl-11C-clozapine in the human brain was studied in five healthy volunteers with positron emission tomography (PET). Four of the volunteers were reexamined after treatment with the dopamine D1 and D2 receptor antagonist flupenthixol, and all five volunteers were reexamined after pretreatment with the serotonin2 receptor antagonist ritanserin. The examinations after flupenthixol and ritanserin treatment were performed on different occasions. In the flupenthixol part of the study, two of the subjects were given an oral dose of 1 mg flupenthixol 2-3 h before the posttreatment study with PET. The other two subjects received 0.5 mg orally three times during the 24 h preceding the posttreatment PET study, with the last dose being administered < or = 4 h before the scan. All five ritanserin-treated subjects followed the same dosing regimen. During the 5 days preceding the posttreatment PET study, they were given a 10-mg tablet of ritanserin in the evening. The last dose was administered 2-1/2 hours before the study. Both flupenthixol and ritanserin pretreatment were associated with decreased binding of N-methyl-11C-clozapine in dorsolateral and medial frontal cortical regions. These results support previous findings that clozapine has affinity for both dopamine D1 and serotonin 5-HT2 receptors in the human frontal cortex. No consistent change of binding was observed in striatal regions following flupenthixol or ritanserin pretreatment. The clinical aspects of this feature are discussed, both with respect to efficacy and side effects.

A fully automated method for separation of L-[methyl-11C]Methionine from metabolites in patient plasma was developed. L-[methyl-11C]Methionine was isolated from plasma by solid-phase extraction (SPE). The radioactivity retained on the SPE column was eluted and injected onto the HPLC system for separation of in vivo formed L-[methyl-11C]methionine radiolabeled metabolites. The yield through the isolation procedure and HPLC analysis was greater than 95% with a precision better than 5% (R.S.D.). The calculated rate of L-[methyl-11C]methionine transport into tumor tissue was markedly different with and without compensation for radiolabeled metabolites in patient plasma.

Study Objectives: To investigate the pharmacokinetics of propofol in combination with epidural anesthesia or with intravenous (IV) alfentanil infusion, and to investigate the clinical feasibility of this anesthetic technique in lower abdominal surgery. Design: Randomized, open clinical study. Setting: Operating theaters and postanesthesia recovery unit at the department of gynecology of a university medical center. Patients: 20 healthy, consenting ASA physical status I and II adult female patients undergoing lower abdominal surgery. Interventions: A total IV anesthetic technique was used in all patients. In Group 1, a continuous infusion of propofol was combined with an epidural block with bupivacaine. Group 2 patients received a combination of propofol and alfentanil infusions. Measurements and Main Results: The pharmacokinetics of propofol are best fitted to a two-compartment open model and were similar in both patient groups. Propofol blood clearance was 29.8 ± 6.51 ml/min/kg, and volume of distribution was 3.60 ± 1.34 L/kg, resulting in a blood elimination half-life of 144 ± 46 minutes. The entire period of anesthesia was stable in the alfentanil group. In the epidural group, the initial period of anesthesia was too light as judged by the autonomic response to tracheal intubation, which also correlated in time to a lower propofol blood concentration than was seen in the alfentanil group. No evidence was found that an epidural block induced a change in propofol kinetics, apart from the lower blood concentration during the initial period of anesthesia. Conclusions: We could not show any statistically significant influence of an epidural blockade on the pharmacokinetic parameters of propofol. Nevertheless, the concentration-time profile changed during infusion, rendering the described infusion regimen, in combination with epidural anesthesia, unsatisfactory for adequate hypnosis. The propofol infusion regimen combined with alfentanil provided immediate and stable blood concentrations that were adequate for surgery.

The present study demonstrates the effects of 6R- l -erythro-5,6,7,8-tetrahydrobiopterin (6R-BH 4 ) on turnover of dopamine and serotonin (5-HT) in rat striatum during continuous infusion of the amino acids tyrosine and tryptophan. By monitoring with microdialysis, it was found that the increase in dopamine and homovanillic acid (HVA) concentrations in rat striatal extracellular fluid (ECF) induced by 6R-BH 4 was further enhanced by the continuous infusion of tyrosine at a relatively low dose (1 μmol/min/kg) as compared with the concentration which saturates tyrosine hydroxylation. This dose of tyrosine alone did not induce the elevation of dopamine and HVA concentrations in ECF. In contrast, though the concentration of 5-HT and 5-HIAA in striatal ECF was gradually increased by tryptophan infusion, 6R-BH 4 had no further effect. Although the higher output of dopamine into ECF was induced by the dialytic perfusion of 6R-BH 4 via the microdialysis probe into striatum, tyrosine infusion had no further effect on dopamine concentration in the dialysates. The in vivo measurement of DOPA accumulation during NSD 1015 perfusion suggests that the enhancement of dopamine concentration in ECF induced by tyrosine infusion and 6R-BH 4 might be attributable to an increase in tyrosine hydroxylase activity in striatum. Tryptophan hydroxylase was also activated by tyrptophan infusion and/or 6R-BH 4 , however, it did not induce an increase in 5-HT concentration in striatal ECF.

Study Objective: To determine the effects of intravenous (IV) 9-amino-1,2,3,4-tetrahydroacridine (THA) on postoperative somnolence in comparison, to its pharmacokinetics. Design: Open-label study of the Pharmacokinetics and effects of THA. Setting: Postoperative intensive care ward at the Department of Neurosurgery, Uppsala University Hospital, Sweden. Patients: Ten neurosurgical patients immediately after their operations were given 30 mg of THA for reversal of postoperative somnolence. Interventions: Plasma concentrations of THA and, in seven cases, the metabolite 1-hydroxy-THA were assayed using high-performance liquid chromatography. The pharmacokinetic data were compared to the degree of sedation and pain relief. After an IV dose of 30 mg THA, plasma concentrations were fit to an open two-or three-compartment model. Measurements and Main Results: The antagonistic effect of THA on sedation occurred immediately following IV administration and lasted 60 to 90 minutes. At a mean plasma THA concentration of 44 ± 18 ng/ml, patients were resedated. There was no obvious relation between analgesia and plasma THA concentrations. Side effects such as nausea, salivation, and lower heart rate (HR) were observed in several patients. Plasma clearance (C1) was high and showed a twofold interindividual variation, with a mean of 2.64 ± 1.17 L/h. Volume of distribution (Vd gamma) varied between 300 and 850 liters, with a mean o f 477 ± 185 liters. The plasma half-lives of rapid and slow distribution, and elimination were 2.1 ± 0.7 minutes, 26 ± 18 minutes, and 133 ± 48 minutes, respectively. Maximum plasma concentrations of 1-hydroxy-THA were 27 to 90 ng/ml in five patients; the concentration was below 1 ng/ml in two other patients. Conclusions: The duration of the effects of THA as an antagonist o f postoperative sedation was only about double that seen previously after the IV administration of physostigmine in a similar group of patients, although the elimination half-life of THA was six times longer than that of physostigmine. A larger dose of THA possibly could have been given to prolong the period o f antagonism of sedation, but the profile of adverse effects seen even at the doses used precluded that option.

Positron emission tomography (PET) is an in vivo autoradiographic technique that determines the radioactive distribution and kinetics of a radiolabelled tracer in a tissue. By choice of tracer, it is possible to study physiological processes in living animals and man non-invasiveley. PET has certain disadvantages such as limited spatial resolution and similtaneous measurement of radiolabelled tracer with the metabolites formed. For an adequate interpretation of the data obtained, complementary techniques such as column liquid chromatography of radioactive composition in blood, plasma, urine and tissue samples have to be used. The prerequisites for any chromatographic technique used for the radioanalysis of substances are speed, high selectivity and high separation efficiency. Examples from PET studies in combination with chromatographic analysis will be given. The utilitzation of l -dopa in the brain constitutes several steps. Analysis by column liquid chromatography of metabolites in plasma and in monkey brain tissue will make it possible to elucidate different utilization processes of the tracer. Kinetic studies of 11 C-labelled neuropeptides such as methionine-enkephalin and substance P revealed high radioactivities in the brain of monkeys. However, simultaneous determination plasma and urine radioactivities using liquid chromatography with radiochemical and photometric detection both indicated that the brain radioactivities emanated to a large extent from 11 C-labelled metabolites formed in vivo . Studies with PET using radiotracers having a rapid and extensive metabolism require complementary techniques in the evaluation. High detection selectivity, by combination of photometric and radiochemical detection and rapid and efficient separation, will make liquid chromatography a most important complement in the analysis.

Presynaptic serotonin imaging in social phobia using [3-11C]-5-hydroxy-L-tryptophan and PET

The electron capture response of twenty-three sulphonamide derivatives has been determined. The detector response was measured after derivatization of the simple sulphonamides with different alkyl and acyl reagents in order to evaluate the structural requirements for a high response towards the electron capture detector. An electron capture response of the order of 10 -15 mole/sec was obtained with derivatives with two functional groups, e.g. , phenyl or carbonyl, in conjugation with the sulphonamide group. The response decreased some 100 times with only one such group present. Sulphonamide derivatives with a high electron capture response showed a decreased response, or were insensitive, towards an increased detector temperature.

Two-phase derivatization with chloroformate esters of tertiary amines that contain a phenol group has been studied. The derivatives were analysed by gas chromatography with sensitive and selective detectors. The N,O-bis derivative of pentazocine was formed using an aqueous phase at pH 9.8, with tetrabutylammonium ion for extraction of the phenolate ion as an ion-pair. The organic phase comprised methylene chloride with trichloroethyl chloroformate or ethyl chloroformate. At pH 10.7 the derivative was hydrolysed by hydroxide ions extracted into the organic phase. Hydrolysis also occurred when more lipophilic counter-ions such as tetrapentyl-ammonium ions were used. Two-phase derivatization could also be achieved with toluene or ethyl acetate as the organic phase, although the reaction rate was considerably slower. The method was also applied to the assay of pentazocine in plasma. The best results were achieved after selective extraction of pentazocine from plasma, two-phase derivatization with ethyl chloroformate and determination by mass spectrometry or thermionic detection. The minimum detectable concentration of pentazocine was 10 ng/ml in a 1-ml plasma sample.

The kinetics of four 11 C-labelled enkephalin peptides: Tyr-Gly-Gly-Phe-Met (Met-enkephalin), Tyr- d -Met-Gly-Phe-Pro-NH 2 (( d -Met 2 ,Pro 5 )-enkephalinamide), Tyr- d -Ala-Gly-Phe-Met-NH 2 (DALA) and Tyr- d -Ala- d -Ala-Phe-Met-NH 2 (TAAFM) all labelled at the methyl group of methionine was studied in the Rhesus monkey. After intravenous administration, the regional kinetics in the head, lungs, liver and kidneys were followed by means of positron emission tomography (PET). The total radioactivity in blood and urine was measured and the composition of 11 C-labelled peptide fragments in plasma in vivo and in vitro was analysed by liquid chromatography. With PET, an increased radioactivity was observed in the brain and pituitary over the 60–90 min investigation period after i.v. injection of the peptides. The highest radioactivities were noted for Met-enkephalin, followed by DALA and d -Met 2 , Pro 5 -enkephalinamide, while very low radioactivities were found for TAAFM. The uptake of Met-enkephalin- and DALA-derived radioactivity was of the same order as has previously been shown for morphine in the brain and considerably higher than that of d -Met 2 ,Pro 5 -enkephalinamide and TAAFM, respectively. A large fraction of the brain radioactivity derived from Met-enkephalin and DALA probably emanated from [ 11 C]methionine as indicated by plasma and urine analysis. Met-Enkephalin was rapidly eliminated from plasma in vitro with an half-life of less than two minutes, whereas DALA was stable suggesting clearance by other tissues than plasma. In conclusion, both Met-enkephalin and DALA, were rapidly hydrolyzed in vivo to [ 11 C]methionine. [ 11 C]Methionine was probably taken up in the brain, as the radioactivity increased with time in different brain regions as measured with PET. d -Met 2 ,Pro 5 -Enkephalinamide and TAAFM were virtually stable in vivo and at least part of the radioactivity observed in the brain may have represented the intact peptide.

The rate of extractive alkylation of 24 structurally related sulphonamides has been studied with respect to the alkylating agent and organic solvent. The reaction rate is given by the time for quantitative alkylation calculated from the observed first-order rate constant with 0.1 M tetrabutylammonium (pH 10) as the aqueous phase. The highest reaction rate were obtained with methyl iodide and pentafluorobenzyl bromide as reagents and methylene chloride and methyl isobutyl ketone as organic solvents. The minimum detectable concentration of the N1-methyl derivatives with electron-capture detection was about 10−15 mole/sec.

Conditions for the extractive alkylation of eight sulphonylurea hypoglycemic drugs have been evaluated. Extractive methylation of the compounds was achieved within 90 min using tetrabutylammonium as counter-ion (0.1 M at pH = 6.9) with 5% methyl iodide in dichloro-methane as organic phase. Mass spectral analysis showed derivatives methylated at the sulphonamide nitrogen. A higher pH or use of tetrapentylammonium as counter-ion caused hydrolysis of the sulphonylureas. The derivatives showed a high electron-capture response with minimum concentrations detectable in the range 1-4 x 10(-16) moles sec-1. Therapeutic plasma concentrations of glipzide and tolbutamide were determined by direct extractive methylation of the compounds from the plasma sample. The glipizide derivative was determined by electron-capture gas chromatography down to about 20 ng/ml in a 0.5-ml plasma sample. The relative standard deviation at the 0.2 microgram/ml level of glipizide was 6% (n = 6). The corresponding figure in the determination of tolbutamide at the 10 microgram/ml level was 3% (n = 10).

Nicotine was subjected to reaction at 90 degrees with trichloroethyl chloroformate in the presence of pyridine to form a carbamate in which the pyrrolidine ring was opened. Upon heat treatment, this carbamate partially formed the corresponding olefin. About 10 pg could be detected with an electron-capture detector and 60 pg with an alkali flame-ionization detector. The extraction was studied with 14C-labelled nicotine. Methylene chloride was suitable for extraction from diluted plasma, whereas toluene containing 5% of heptafluorobutanol was used in a re-extraction step and also as the chloroformate reaction medium. Due to a nicotine blank the limit for quantitative determinations was 10 ng/ml in plasma (sample volume 1 ml). N-n-Propylnornicotine was used as an internal standard. The precision at the 30 ng/ml level was +/- 8.8% (n = 7).

The pharmacokinetics of azathioprine (AZA) and 6-mercaptopurine (6-MP) was studied in uremic patients after 100 mg AZA intravenously (fifteen patients) and orally (eight patients). 6-MP was analysed with gas chromatography mass spectrometry following extractive alkylation. AZA was determined indirectly assuming quantitative conversion to 6-MP in whole blood. The plasma concentration of AZA fell rapidly after i.v. administration. The mean half-time of elimination for the first rapid phase ( t 1 2 α was 6.1 min (S.D. ± 4.1) and for the terminal phase ( t 1 2 β ) 50 min (± 31). The total plasma clearance (C1) was 6.91./min (± 3.0). AZA was rapidly converted to 6-MP in vitro, and maximal plasma concentrations of 6-MP were found as early as 5 min after i.v. injection of AZA. The mean t 1 2 α was 4.6 min (± 2.2), t 1 2 β 74 min (± 58) and C1 8.01/min (± 5.8). The plasma levels of both AZA and 6-MP were either low or undetectable 4–6 h after dose. In erythrocytes AZA levels were low or undetectable indicating rapid conversion to 6-MP in these cells. 6-MP concentration - time curve in erythrocytes was similar to that in plasma, except for a somewhat slower terminal phase of elimination. Oral administration of AZA generated flat plasma curves for AZA and 6-MP. The area under the concentration - time curve (AUC) was considerably smaller than after i.v. administration, 18 and 41% for AZA and 6-MP, respectively. There seems to be little danger of accumulation of AZA/6-MP in uremia. We also studied inhibition of Leucoagglutin (LA) stimulated lymphocyte proliferation by patient plasma at different times in six of the patients following AZA i.v. Sera drawn at 5, 10 and 30 min significantly inhibited the LA-induced proliferation, with an estimated minimum effective concentration of 6-MP in the cultures of about 0.02–0.04 μM. This suggests the possibility of a therapeutic effect even of the low plasma levels of 6-MP obtained after AZA orally. The combined use of sensitive pharmacokinetic and immunological assays as described should be useful in studying the relationship between plasma levels of AZA/6-MP and their immunosuppressive effect and toxicity.

Pethidine (meperidine) is determined in plasma by electron capture gas chromatography after derivatization with trichloroethyl chloroformate. The analytical procedure involves extraction of pethidine and the internal standard from plasma and their separation from metabolites by partition chromatography. After purification of the eluate, the derivatization is accomplished with trichloroethyl chloroformate in the presence of anhydrous sodium carbonate. The reaction mixture is further purified with methanolic alkali before gas chromatographic analysis. Optimum conditions for extraction and derivatization, as well as the sensitivity and selectivity of the method are discussed. Owing to the high sensitivity the pethidine levels are determined in 0.1 ml of plasma. The smallest amount of pethidine determined by the method was 100 ng/ml. The relative standard deviation at the 50-ng level of pethidine added to 0.1 ml of plasma was 5.8% (n = 8).

An analytical procedure was developed for the determination of 6-mercaptopurine in plasma. Owing to the polar character and low plasma concentration of the compound, extraction and derivatization was carried out directly from the plasma sample by extractive alkylation. Determination was made using gas chromatography-mass spectrometry with multiple-ion detection. Conditions with respect to the rate of formation and the stability of the derivative formed in the extractive alkylation step were evaluated. The selectively of the method to azathioprine and to metabolites was thoroughly investigated. No 6-mercaptopurine was formed from azathioprine added to water or plasma and run through the method. The method enables the detection of 2 ng of 6 mercaptopurine in a 1.0-ml plasma sample. Quantitative determinations were done down to 10 ng/ml 6 mercaptopurine in plasma.

A selective procedure is described for the determination of amitriptyline in plasma. The method involves extraction, separation of amitriptyline from its metabolites and subsequent oxidation by ceric sulphate in 5.4 M sulphuric acid. The oxidation product, anthraquinone, is determined by means of electron-capture gas chromatography. The metabolites were separated by a column chromatographic extraction technique. The choice of oxidation reagent, optimum conditions for the oxidation, and the electron-capture properties of anthraquinone are discussed. The method can be used to determine down to 2 ng of amitriptyline in a plasma sample; the relative standard deviation at the 50-ng level was 4.0% (n = 8). The levels of amitriptyline found in a series of plasma samples are compared with those obtained by gas chromatography with use of nitrogen-specific detection; the two techniques gave coincident results.