P-glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Main Active Heroin Metabolites 6-monoacetylmorphine (6-MAM) and Morphine in Mice.

BACKGROUND & PURPOSE: Heroin (diacetylmorphine; diamorphine) is a highly addictive opioid prodrug. Heroin prescription is possible in some countries for chronic, treatment-refractory opioid-dependent patients and as a potent analgesic for specific indications. We aimed to study the pharmacokinetic interactions of heroin and its main pharmacodynamically active metabolites, 6-monoacetylmorphine (6-MAM) and morphine, with the multidrug efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2 using wild-type, Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice. METHODS & RESULTS: Upon subcutaneous (s.c.) heroin administration, its blood levels decreased quickly, making it challenging to detect heroin even shortly after dosing. 6-MAM was the predominant active metabolite present in blood and most tissues. At 10 and 30 min after heroin administration, 6-MAM and morphine brain accumulation were increased about 2-fold when mouse (m)Abcb1a/1b and mAbcg2 were ablated. Fifteen minutes after direct s.c. administration of an equimolar dose of 6-MAM, we observed good intrinsic brain penetration of 6-MAM in wild-type mice. Still, mAbcb1 limited brain accumulation of 6-MAM and morphine without affecting their blood exposure, and possibly mediated their direct intestinal excretion. A minor contribution of mAbcg2 to these effects could not be excluded. CONCLUSIONS: We show that mAbcb1a/1b can limit 6-MAM and morphine brain exposure. Pharmacodynamic behavioral/postural observations, while non-quantitative, supported moderately increased brain levels of 6-MAM and morphine in the knockout mouse strains. Variation in ABCB1 activity due to genetic polymorphisms or environmental factors (e.g., drug interactions) might affect 6-MAM/morphine exposure in individuals, but only to a limited extent.

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.

Metabolism of trabectedin (ET-743, Yondelis) in patients with advanced cancer.

PURPOSE: Trabectedin (ET-743, Yondelis) is a novel anti-cancer drug currently undergoing phase II-III evaluation, that has shown remarkable activity in pre-treated patients with soft tissue sarcoma. Despite extensive pharmacokinetic studies, the human disposition and metabolism of trabectedin remain largely unknown. We aimed to determine the metabolic profile of trabectedin and to identify its metabolites in humans. METHODS: We analysed urine and faeces (the major excretory route) from eight cancer patients after a 3 or 24 h intravenous administration of [14C]trabectedin. Using liquid chromatography with tandem quadrupole mass spectrometric detection (LC-MS/MS) and radiochromatography with off-line radioactivity detection by liquid scintillation counting (LC-LSC), we characterised the metabolic profile in 0-24 h urine and 0-120 h faeces. RESULTS: By radiochromatography, a large number of trabectedin metabolites were detected. Incubation with beta-glucuronidase indicated the presence of a glucuronide metabolite in urine. Trabectedin, ET-745, ET-759A, ETM-259, ETM-217 (all available as reference compounds) and a proposed new metabolite coined ET-731 were detected using LC-MS/MS. The inter-individual differences in radiochromatographic profiles were small and did not correlate with polymorphisms in drug-metabolising enzymes (CYP2C9, 2C19, 2D6, 2E1, 3A4, GST-M1, P1, T1 and UGT1A1 2B15) as determined by genotyping. CONCLUSIONS: Trabectedin is metabolically converted to a large number of compounds that are excreted in both urine and faeces. In urine and faeces we have confirmed the presence of trabectedin, ET-745, ET-759A, ETM-259, ETM-217 and ETM-204. In addition we have identified a putative new metabolite designated ET-731. Future studies should be aimed at further identification of possible metabolites and assessment of their activity.

Phase I and pharmacokinetic study of combined treatment with perifosine and radiation in patients with advanced solid tumours.

PURPOSE: Perifosine is an orally applicable, membrane-targeted alkylphosphocholine analogue with antitumour activity and radiosensitising properties in preclinical models. The purpose of this phase I study was to determine the feasibility and tolerability of concurrent daily perifosine and radiation in patients with advanced cancer. PATIENTS AND METHODS: Starting dose of perifosine was 50 mg/day; dose escalation was in steps of 50mg. Daily administration commenced 2 days before radiotherapy and was continued throughout the radiation treatment. At least three patients were entered at each dose level; at the 150 mg/day level 10 patients were included. Pharmacokinetic sampling was performed weekly pre-dosing. Twenty-one patients were entered. Tumour types included NSCLC (n=17), prostate, oesophageal, colon and bladder cancer. Most patients (16/21) had received prior chemotherapy; none radiotherapy. Median number of daily perifosine administrations was 31 (range 24-53). Mean radiation dose (BED(10)) was 59.8 Gy (range 50.7-87.5 Gy in 13-28 fractions). RESULTS: Major drug-related toxicities according to CTC criteria were nausea in 57%, fatigue in 48%, vomiting in 38%, diarrhoea in 38% and anorexia in 19%. No bone marrow toxicity was observed. DLT (nausea/vomiting) was encountered in two of five patients at the 200mg/day dose level. Dose-dependent steady-state plasma levels were reached after 1 week. Major radiotherapy-related acute toxicity consisted of dysphagia in 38% and pneumonitis in 29%. CONCLUSION: Perifosine can be safely combined with fractionated radiotherapy. A dosage of 150 mg/day, to be started at least 1 week prior to radiotherapy, is recommended for phase II evaluation.

Quantitative analysis of EO9 (apaziquone) and its metabolite EO5a in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray tandem mass spectrometry.

A sensitive and specific LC-MS/MS assay for the quantitative determination of EO9 and its metabolite EO5a is presented. A 200-microl human plasma aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 ml ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate-methanol (7 : 3, v/v) and 25 microl-volumes were injected into the HPLC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide-methanol (gradient system)). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 5 to 2500 ng/ml for EO9 and from 10 to 2500 ng/ml EO5a using 200 microl of human plasma samples. Validation results demonstrate that EO9 and EO5a concentrations can be accurately and precisely quantified in human plasma. This assay will be used to support clinical pharmacologic studies with EO9.

Pharmacokinetics and pharmacodynamics of high doses of pharmaceutically prepared heroin, by intravenous or by inhalation route in opioid-dependent patients.

A pharmacokinetic-pharmacodynamic study was performed in opioid-dependent patients in the Netherlands, who were currently treated with high doses of pharmaceutically prepared heroin on medical prescription. Besides intravenous heroin, heroin was prescribed for inhalation by "chasing the dragon" method. In this technique, heroin base is heated on aluminium foil, and heroin vapours are inhaled into the lungs. Not much is known about the pharmacokinetics profile and bioavailability of this specific administration method. Therefore, a study was performed on pharmacokinetics and pharmacodynamics of heroin inhalation and intravenous use. Eleven patients who injected heroin and 9 patients who inhaled heroin entered the study. They were on steady-state heroin treatment for at least 12 months. For safety reasons, there was no crossing-over between heroin injection or inhalation. In a double-blind randomised study, 67-100-150% of the regular heroin maintenance dose was administered to each patient. Maximal single heroin dose was 450 mg. Plasma concentrations of heroin and its metabolites 6-monoacetylmorphine, morphine and morphine-glucuronides were analysed using LC-MS-MS. Blood pressure, heart rate, skin temperature and reaction time were assessed. Furthermore, visual analogue scales regarding craving and appreciation of heroin effect were scored by the subjects. Both in inhaling and injecting patients, the areas under curve of heroin and all measured metabolites were linearly related to heroin dose. Mean C(max) of heroin and its metabolites were 2-6 times lower after inhalation, than after intravenous injection. Bioavailability (F) of heroin inhalation was estimated as 52% (95% CI 44-61%). Heroin was rapidly cleared from plasma. Cl/F was 930 l/hr (95% CI 799-1061 l/hr) after intravenous administration, and 1939 l/hr (95% CI 1661-2217 l/hr) after inhalation. Heroin Cl and Vd were correlated to body weight (R(2) 15-19%). Morphine-glucuronides levels were inversely related to creatinine clearance. After heroin administration, the reaction time was significantly prolonged with 28+/-5.3 msec. in injecting and 13+/-4.9 msec. in inhaling patients. Cardiovascular changes were only mild after heroin administration. Craving-scores declined immediately after heroin administration in both administration groups. Subjective heroin effect was rated more positively in heroin inhaling than in injecting patients, despite the lower C(max) levels following heroin inhalation. In both groups, in this blinded study heroin dose increments were more appreciated than dose reductions. Increments of 50% of the regular heroin dose did not cause any serious side effect.

Screening for illicit heroin use in patients in a heroin-assisted treatment program.

The aim of this study was to investigate the use of illicit heroin among patients in a heroin-assisted treatment program. In this program, pharmaceutical-grade heroin was administered to heroin-addicted patients. Monitoring of illicit heroin use was considered important for the evaluation of this treatment program. Acetylcodeine and codeine, common adulterants of "street" heroin, were used as markers for illicit heroin. A liquid chromatography method with tandem mass spectrometric detection (LC-MS-MS) was developed, for quantitative analysis of heroin and methadone, their metabolites, and the simultaneous detection of acetylcodeine. One-hundred patients in a heroin-assisted treatment program were screened for acetylcodeine in plasma. Furthermore, patients were interviewed about illicit heroin use, and they were tested for alcohol and cocaine use. In plasma samples of 16% of the patients, acetylcodeine was detected. Overall agreement between self-report and plasma samples was 95% (kappa: 0.81). Patients who tested positive for acetylcodeine had visited the outpatients' clinics significantly less frequently than the patients who tested negative. Alcohol and cocaine use was more common in patients who tested positive for acetylcodeine. Illicit heroin use was observed in a limited percentage of patients. Overall agreement between self-report and markers of illicit heroin use was good.

Analysis of diacetylmorphine, caffeine, and degradation products after volatilization of pharmaceutical heroin for inhalation.

Pharmaceutical smokable heroin was developed for a clinical trial on medical co-prescription of heroin and methadone. This product, consisting of 75% w/w diacetylmorphine base and 25% w/w caffeine anhydrate, was intended for use via "chasing the dragon", that is, inhalation after volatilization. This procedure involves heating the powder mixture, which may lead to formation of degradation products that could subsequently be inhaled. We developed a method that used a high-performance liquid chromatography system that was compatible with photodiode-array detection and mass spectrometric detection to separate diacetylmorphine- and caffeine-related compounds in a wide polarity range for analysis of the vapor. This method was used to analyze the contents of the plastic drinking straws that were used by patients to inhale the vapors from pharmaceutical heroin used via chasing the dragon, which were considered to be representative of the vapors the patients inhaled. They contained primarily unchanged diacetylmorphine, its main metabolite 6-acetylmorphine, caffeine, and some morphine. Several unidentified peaks were observed in the straw chromatograms. Chemical structures were proposed for nine degradation products: morphine derivatives with different substitution patterns of the C(3), C(6), and/or N(17) positions, which comprised 0.4-9.7% of the straw sample residue weight. Activity and toxicity of most of these compounds are unknown and require further investigation.

Metabolism of docetaxel in mice.

Previous studies have shown by quantification of the parent drug and the known metabolites M-1, M-2, M-3 and M-4 that the mass balance of docetaxel in mice and humans is not complete. We therefore used reversed-phase high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection and tandem mass spectrometry to trace and identify putative metabolites in the feces and bile of mice injected intravenously with docetaxel. HPLC-PDA revealed two metabolic products in the feces and more than ten potential new metabolites in the bile. Mass spectrometry was performed on docetaxel reference compound, on the known metabolites M-1, M-2, M-3 and M-4, and on HPLC eluate fractions containing metabolic products, six fractions originating from the bile and two from the feces. The mass spectra of the most abundant unknown metabolite in the bile and the feces were identical, and indicated that this structure contained a carboxyl moiety at the tert-butyl group. Under the conditions of storage this product degraded to metabolite M-4. All other unknown metabolites found in the bile samples were oxidized products, with the oxidations in both the C-13 side chain and the baccatin structure, the latter being a new finding.

Pharmacokinetic comparison of two methods of heroin smoking: 'chasing the dragon' versus the use of a heating device.

In preparation for a trial on co-prescription of inhalable heroin and methadone, two methods for inhalation of heroin/caffeine tablets were compared: the commonly used method of 'chasing the dragon' and a standardised procedure for inhalation of volatilised heroin, using a heating device. Five male addicts inhaled a tablet of smokable heroin daily for 5 days, alternating the inhalation method. Plasma concentrations of heroin, 6-acetylmorphine, morphine and morphine-3- and -6-glucuronide were determined using a liquid chromatography method with tandem mass spectrometric detection. The exposure to heroin and its metabolites (expressed as areas under the concentration-time curve) was significantly lower after smoking via the heating device than after 'chasing the dragon': heroin 80% and 6-acetylmorphine 73% lower (p < 0.05). Maximal concentrations of heroin and 6-acetylmorphine were also 80% and 70% lower (p < 0.05) after using the heating device. 'Chasing the dragon' is a more efficient inhalation method than inhalation via the heating device.

The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry.

For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 degrees C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5-500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (< or =20% at lower limit of quantification (LLQ) and < or =15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.

LC-UV method development and validation for the investigational anticancer agent imexon and identification of its degradation products.

Imexon (4-imino-1,3-diazabicyclo[3,1,0]-hexan-2-one) is a member of the class of 2-cyanoaziridine derivatives, which have been of interest as immunomodulators and anticancer agents since the late 1970s. The pharmaceutical development of imexon necessitated the availability of an assay for the quantification and purity determination of imexon active pharmaceutical ingredient (API) and the drug in its pharmaceutical dosage form. A liquid chromatographic (LC) method with ultraviolet (UV) detection was developed, using a reverse phase column with phosphate buffer (pH 6; 50 mM) as mobile phase and UV detection at 230 nm. Although retention capacity for imexon was small (capacity factor of 0.5), the method was found to be linear over the concentration range of interest of 1.0-25 microg/mL, precise, accurate, and stability-indicating. Moreover, the use of LC-mass spectrometry (MS) and on-line photodiode array (PDA) detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API. The degradation products, including chloro- and hydroxy-derivatised products were shown to arise from nucleophilic reactions with the activated aziridine moiety of imexon. The developed LC-UV method was found suitable for the pharmaceutical quality control of imexon API and the drug in its pharmaceutical dosage form.

Development and validation of an LC-UV method for the quantification and purity determination of the novel anticancer agent C1311 and its pharmaceutical dosage form.

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. The pharmaceutical development of C1311 necessitated the availability of an assay for the quantification and purity determination of C1311 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase liquid chromatographic method (RP-LC) with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with phosphate buffer (60 mM; pH 3 with 1 M citric acid)-acetonitrile-triethylamine (83:17:0.05, v/v/v) as the mobile phase and UV-detection at 280 nm. The method was found to be linear over a concentration range of 2.50-100 microg/mL, precise and accurate. Accelerated stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indicating capacity. Moreover, the use of LC-MS and on-line photo diode array detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API and more abundantly in an impure lot of API.

Deuterodiacetylmorphine as a marker for use of illicit heroin by addicts in a heroin-assisted treatment program.

In preparation for a treatment program concerning the medical coprescription of heroin and methadone to treatment-resistant addicts in the Netherlands, we studied a novel strategy for monitoring co-use of illicit (nonprescribed) heroin. A deuterated analogue of heroin was added (1:20) to pharmaceutical, smokable heroin (a powder mixture of 75% w/w diacetylmorphine base and 25% w/w caffeine anhydrate), to be used by inhalation after volatilization ("chasing the dragon"). Plasma and urine samples were collected from nine male patients who had used pharmaceutical, smokable heroin during a four-day stay in a closed clinical research unit, and these samples were analyzed by liquid chromatography coupled with tandem mass spectrometry. Ratios of deuterated and undeuterated diacetylmorphine and 6-acetylmorphine (MAM/MAM-d3) in plasma and urine were calculated from peak areas of these substances in the respective chromatograms. The MAM/MAM-d3 ratios in plasma and urine were normally distributed (with small standard deviations) and independent from concentrations of 6-acetylmorphine and from time after use of pharmaceutical heroin. A MAM/MAM-d3 ratio in urine above 32.8 was considered indicative of co-use of illicit heroin, and this value was associated with a false-positive rate of only 1% (95% confidence interval: -1 to 3%). The MAM/MAM-d3 ratio was detectable in urine for 4-9.5 h after use of pharmaceutical, smokable heroin. Addition of stable, isotopically labelled heroin to pharmaceutical, smokable heroin is considered to be a feasible strategy for the detection of co-use of illicit heroin by patients in heroin-assisted treatment.

Development of an LC-MS/MS assay for the quantitative determination of the intracellular 5-fluorouracil nucleotides responsible for the anticancer effect of 5-fluorouracil.

5-Fluorouracil (5-FU) and its oral prodrug capecitabine are among the most widely used chemotherapeutics. For cytotoxic activity, 5-FU requires cellular uptake and intracellular metabolic activation. Three intracellular formed metabolites are responsible for the antineoplastic effect of 5-FU: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). In this paper, we describe the development of an LC-MS/MS assay for quantification of these active 5-FU nucleotides in peripheral blood mononuclear cells (PBMCs). Because the intracellular 5-FU nucleotide concentrations were very low, maximization of the release from the cell matrix and minimization of interference were critical factors. Therefore, a series of experiments was performed to select the best method for cell lysis and nucleotide extraction. Chromatography was optimized to obtain separation from endogenous nucleotides, and the effect of different cell numbers was examined. The assay was validated for the following concentration ranges in PBMC lysate: 0.488-19.9 nM for FUTP, 1.66-67.7 nM for FdUTP and 0.748-30.7 nM for FdUMP. Accuracies were between -2.2 and 7.0% deviation for all analytes, and the coefficient of variation values were ≤ 4.9%. The assay was successfully applied to quantify 5-FU nucleotides in PBMC samples from patients treated with capecitabine and patients receiving 5-FU intravenously. FUTP amounts up to 3054 fmol/10(6) PBMCs and FdUMP levels up to 169 fmol/10(6) PBMCs were measured. The FdUTP concentrations were below the lower limit of quantification. To our knowledge, this is the first time that 5-FU nucleotides were quantified in cells from patients treated with 5-FU or capecitabine without using a radiolabel.

Simultaneous quantification of cyclophosphamide, 4-hydroxycyclophosphamide, N,N',N"-triethylenethiophosphoramide (thiotepa) and N,N',N"-triethylenephosphoramide (tepa) in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

The alkylating agents cyclophosphamide (CP) and N, N', N"-triethylenethiophosphoramide (thiotepa) are often co-administered in high-dose chemotherapy regimens. Since these regimens can be complicated by the occurrence of severe and sometimes life-threatening toxicities, pharmacokinetically guided administration of these compounds, to reduce variability in exposure, may lead to improved tolerability. For rapid dose adaptations during a chemotherapy course, we have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of CP, thiotepa and their respective active metabolites 4-hydroxycyclophosphamide (4OHCP) and N, N', N"-triethylenephosphoramide (tepa) in plasma. Because of the instability of 4OHCP in plasma, the compound is derivatized with semicarbazide (SCZ) immediately after sample collection and quantified as 4OHCP-SCZ. Sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 microl of plasma. Chromatographic separation was performed on an Zorbax Extend C18 column (150 x 2.1 mm i.d., particle size 5 microm), with a quick gradient using 1 mM ammonia solution and acetonitrile, at a flow-rate of 0.4 ml min(-1). The analytical run time was 10 min. The triple quadrupole mass spectrometer was operating in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 200-40,000 ng ml(-1) for CP, 50-5000 ng ml(-1) for 4OHCP-SCZ and 5-2500 ng ml(-1) for thiotepa and tepa, using 100 microl of human plasma. These dynamic concentration ranges proved to be relevant in daily practice. Hexamethylphosphoramide was used as an internal standard. The coefficients of variation were <12% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/- 15%). This robust and rapid LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring of CP, thiotepa and their metabolites in our hospital.

2-Hydroxypropyl-beta-cyclodextrin extracts 2-phenylphenol from silicone tubing.

Cyclodextrins are capable to solubilise lipophilic drugs via (partial) inclusion in their lipophilic cavity. This, however, also provides the potential for the extraction of small molecules from production materials. In the present study, the potency of the commercially available and used cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) to extract the preservative 2-phenylphenol (2-PP) from platinum cured silicone tubing was tested. The presence of 2-PP was structurally confirmed with HPLC-UV and LC/MS/MS in HPbetaCD solutions after incubation with platinum cured silicone tubing. HPbetaCD concentration and prior tubing sterilisation were found not to influence the levels of 2-PP extracted. Interestingly, extraction to ethanol was 15-fold higher than observed for HPbetaCD solutions. 2-PP was extracted from silicone tubing during routine manufacture of a blank dosage form formulated with only HPbetaCD, resulting in detectable levels of 2-PP in the final product. In a freeze-dried dosage form containing HPbetaCD and an active pharmaceutical ingredient (exhibiting a stability constant for HPbetaCD/drug of 1045 L/mol), on the other hand, 2-PP was undetectable.

Quantitative determination of azacitidine triphosphate in peripheral blood mononuclear cells using liquid chromatography coupled with high-resolution mass spectrometry.

Azacitidine is a cytidine analog used in the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia and acute myeloid leukemia. The pharmacological effect of azacitidine arises after incorporation into the DNA and RNA. To this end, the drug first has to be converted into its triphosphate forms. This paper describes the development of an assay for quantitative determination of azacitidine triphosphate (aza-CTP) in peripheral blood mononuclear cells (PBMCs). To quantify aza-CTP, separation from the endogenous nucleotides cytidine triphosphate (CTP) and uridine triphosphate (UTP) is required. This was a challenge as the structures of these nucleotides are highly similar and the monoisotopic molecular masses of aza-CTP, UTP and the naturally occurring [(13)C]- and [(15)N]-isotopes of CTP differ less than 0.02 Da. Efforts to select a specific MS(2)-fragment for aza-CTP using a triple quadrupole mass spectrometer remained without success. Therefore, we investigated the feasibility to separate these highly resembling nucleotides based on accurate mass spectrometry using a linear trap quadrupole (LTQ) coupled with an Orbitrap. The LTQ-Orbitrap was able to differentiate between aza-CTP and the endogenous nucleotides UTP and [(13)C]-CTP. There was no baseline resolution between aza-CTP and [(15)N]-CTP, but the [(15)N]-CTP interference was low. For quantification, extracted ion chromatograms were obtained for the accurate m/z window of the aza-CTP product ion. The assay was able to determine aza-CTP concentrations in PBMC lysate from 40.7 to 281 nM. Assuming that an average cell suspension extracted from 16 mL blood contains 10 to 42 million PBMCs per mL, this range corresponds with 2.58/10.9-17.8/74.9 pmol aza-CTP per million PBMCs. Intra-assay accuracies were between -1.1 and 9.5% deviation and coefficient of variation values were ≤13.2%. The assay was successfully applied to quantify aza-CTP in samples from two patients treated with azacitidine. Aza-CTP concentrations up to 19.0 pmol per million PBMCs were measured. This is the first time that aza-CTP concentrations were quantified in PBMCs from patients treated with azacitidine.

A sensitive combined assay for the quantification of paclitaxel, docetaxel and ritonavir in human plasma using liquid chromatography coupled with tandem mass spectrometry.

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human plasma is described. The drugs were extracted from 200 µL human plasma using liquid-liquid extraction with tertiar-butylmethylether, followed by high performance liquid chromatography analysis using 10 mM ammonium hydroxide pH 10:methanol (3:7, v/v) as mobile phase. Chromatographic separation was obtained using a Zorbax Extend C(18) column. Labelled analogues of the analytes are used as internal standards. For detection, positive ionization electrospray tandem mass spectrometry was used. Method development including optimisation of the mass transitions and response, mobile phase optimisation and column selection are discussed. The method was validated according to FDA guidelines and the principles of Good Laboratory Practice (GLP). The validated range was 0.5-500 ng/mL for paclitaxel and docetaxel and 2-2000 ng/mL for ritonavir. For quantification, quadratic calibration curves were used (r(2)>0.99). The total runtime of the method is 9 min and the assay combines analytes with differences in ionisation and desired concentration range. Inter-assay accuracy and precision were tested at four concentration levels and were within 10% and less than 10%, respectively, for all analytes. Carry-over was less than 6% and endogenous interferences or interferences between analytes and internal standards were less than 20% of the response at the lower limit of quantification level. The matrix factor and recovery were determined at low, mid and high concentration levels. The matrix factor was around 1 for all analytes and total recovery between 77.5 and 104%. Stability was investigated in stock solutions, human plasma, dry extracts, final extracts and during 3 freeze/thaw cycles. The described method was successfully applied in clinical studies with oral administration of docetaxel or paclitaxel in combination with ritonavir.

Lethal morphine intoxication in a patient with a sickle cell crisis and renal impairment: case report and a review of the literature.

Morphine-6-glucuronide, the active metabolite of morphine, and to a lesser extent morphine itself are known to accumulate in patients with renal failure. A number of cases on non-lethal morphine toxicity in patients with renal impairment report high plasma concentrations of morphine-6-glucuronide, suggesting that this metabolite achieves sufficiently high brain concentrations to cause long-lasting respiratory depression, despite its poor central nervous system penetration. We report a lethal morphine intoxication in a 61-year-old man with sickle cell disease and renal impairment, and we measured concentrations of morphine and morphine-6-glucuronide in blood, brain and cerebrospinal fluid. There were no measurable concentrations of morphine-6-glucuronide in cerebrospinal fluid or brain tissue, despite high blood concentrations. In contrast, the relatively high morphine concentration in the brain suggests that morphine itself was responsible for the cardiorespiratory arrest in this patient. Given the fatal outcome, we recommend to avoid repeated or continuous morphine administration in renal failure.