Journal of Chromatography B (v.772, #2)

News Section (N1-N3).

A simple restricted-access media (RAM) HPLC method for simultaneous determination of the lactone and carboxylate forms of 10-hydroxycamptothecin (HCPT) in human serum was established. Using a RAM Hisep analytical column, serum samples were directly injected into the HPLC system. The eluted peaks of two forms of HCPT were monitored with a fluorescence detector. The separation was completed in 17 min. The linear range was 20–1000 ng/ml, intra-day and inter-day variations being less than 5%. The kinetic equation was introduced according to the analytical results. The equation shows that the course of the HCPT lactone form converting to carboxylate form in human serum at 4 °C is a first-order kinetic course. The concentration of each form at the moment of sampling was calculated by extrapolation.
Keywords: 10-Hydroxycamptothecin;

Clomipramine (CMI) is a typical tricyclic antidepressant with a wide clinical spectrum, being used in major depressive, panic and obsessive-compulsive disorders. The relationship between clinical response and plasma levels of clomipramine and its N-desmethylated (N-desmethylclomipramine, DMCMI) and hydroxy-metabolites remains unclear. In particular, limited information is available on the correlation with clinical response in patients with obsessive-compulsive disorder (OCD). This study describes a new sensitive method to simultaneously determine CMI and its major N-desmethylated and hydroxy-metabolites present in human plasma by HPLC with a UV detector. After a solid-phase extraction from plasma (Isolute C2 columns) the separation of the compounds was performed on a Lichrospher CN column (250×4 mm, 5 μm with a 2-cm pre-column) by an eluent consisting of 10 mM K2HPO4–acetonitrile–methanol (35:25:40 v/v/v) at a flow of 1.5 ml/min. UV detector was set at 214 nm. The lower limit of quantification for all the analytes was at least 5 ng/ml. The coefficients of variation ranged between 2.0 and 4.9% with recovery rates between 97.0 and 100.3%. Linear regression analyses showed correlation coefficients between 0.98 and 0.99. This method is simple, fast and reliable with good specificity and sensitivity. Solid phase extraction is efficient and rapid, allowing the extraction of several plasma samples on the same day and may therefore be usefully and realistically applied in the clinical context. We thus investigated the relevance of plasma levels of CMI and its metabolites as a predictor of clinical outcome in a group of 15 patients with OCD.
Keywords: Clomipramine; Desmethylclomipramine; Hydroxyclomipramine;

Detection of anabolic steroids in animal urine samples is currently performed with GC–MS in our lab. However we found that the detection of 17α-trenbolone (17α-TbOH), 4-chloroandrost-4-ene-3,17-dion (CLAD), 16-β-OH-stanozolol (16OHstan) and α- and β-boldenone (α-Bol, β-Bol) was very difficult, if not impossible. Therefore a sensitive, specific and selective qualitative multi-analyte LC–MS–MS method was developed. The LC separation was achieved by using a Symmetry® C18 column and methanol–water–formic acid (54.7–44.7–0.6) as a mobile phase at a flow-rate of 0.3 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode with positive electrospray interface. Validation of the method was done according to draft SANCO/1805/2000 Rev.1 and a CCβ smaller then 1 ng/ml was obtained for each compound.
Keywords: Steroids, anabolic;

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC–MS–MS) was developed and validated for the quantification of rosuvastatin (Crestor™) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1–30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than ±15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both −20 and −70 °C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.
Keywords: Rosuvastatin; HMG-CoA reductase inhibitor;

A stable isotope dilution liquid chromatography–tandem mass spectrometric (LC–MS–MS) method for the determination of plasma 1α-hydroxyvitamin D3 [1α(OH)D3] has been developed. The method employed derivatization, the reaction with 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione and acetylation, which significantly improved the ionization efficiency of 1α(OH)D3 with a detection limit of 6.3 fmol per injection. The plasma 1α(OH)D3 was extracted with acetonitrile, purified with disposable cartridges, derivatized and subjected to LC–MS–MS analysis using atmospheric pressure chemical ionization. The intra- and inter-assay coefficients of variation were below 10.6 and 4.7%, respectively, and the analytical recovery of 1α(OH)D3 was quantitative. The limit of quantitation was 25 pg/ml for a 1.0-ml plasma aliquot. The application of the developed method to the sample of a volunteer orally given 1α(OH)D3 was also described.
Keywords: 1α-Hydroxyvitamin D3;

A validated method for the quantification of Δ9-tetrahydrocannabinol (THC) and its main metabolites 11-hydroxy-tetrahydrocannabinol (OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) in serum is presented. The substances were isolated by solid-phase extraction, derivatised by methylation, and analysed by means of GC–MS in the selected ion monitoring mode. Quantitation was achieved by the addition of deuterated analogues as internal standards. The method was linear up to 10 ng/ml for THC and OH-THC, and up to 50 ng/ml for THC-COOH. The limits of quantification were 0.62 ng/ml for THC, 0.68 ng/ml for OH-THC and 3.35 ng/ml for THC-COOH. The limits of detection for the least intensive ions were 0.52 ng/ml for THC, 0.49 ng/ml for OH-THC and 0.65 ng/ml for THC-COOH. The method was validated according to the requirements of the Journal of Chromatography B. The method has been routinely used on samples from drivers suspected of “driving under the influence”. In addition to the forensic application, a cross-validation was carried out by applying the method developed for serum to human liver microsomal preparation samples.
Keywords: Δ9-Tetrahydrocannabinol; 11-Hydroxy-Δ9-tetrahydrocannabinol; 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol;

The analysis of solvent residues trapped into crystals of illicit drugs provides useful evidence for monitoring current use trend in the chemical underground, and is also a suitable tool to achieve the complete chemical characterisation of street drugs for comparative examination of separate specimens. This paper describes a method developed in order to perform simultaneous qualitative and quantitative analysis of solvent residues in cocaine samples. The method is based on GC–MS analysis of solvents after their extraction/concentration from drug matrices accomplished by solid-phase microextraction (SPME) in static head space. The proposed method has been used to detect residues of solvents in 47 illicit street cocaine samples. Quantitative analyses were carried out only for the solvents identified at concentration values higher than 1 ppm. Statistical evaluation of our results allowed us to group the illicit samples into various classes according to different kinds of residual solvent, in connection with different clandestine manufacturing processes used to prepare illicit cocaine.
Keywords: Cocaine;

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065 by Feng Bai; Mark N Kirstein; Suzan K Hanna; Clinton F Stewart (257-265).
A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 °C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E 1=200 mV and E 2=600 mV; guard cell: E G=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 μM. The lower limit of quantitation of WR1065 was 0.25 μM. The within- and between-day precisions were ≤4.3% and ≤6.0% for amifostine, ≤4.4% and ≤3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.
Keywords: Amifostine; WR1065;

A highly sensitive, yet simple, isocratic high-performance liquid chromatographic (HPLC) assay with electrochemical detection (ED) for the determination of extracellular dopamine (DA) in brain microdialysates is presented. The method makes possible the detection of less than 100 pM (less than 1 fmol on column) and the quantitation of 200 pM (2 fmol on column) of DA with the use of a narrow-bore rather than capillary or microbore column. Analysis is feasible within an 11-min run-time, and thus is suitable for the relatively short sampling intervals used in microdialysis experiments. In the calibration range of 0.2 to 10 nM, the method has excellent linearity and precision, with intra-day relative standard deviations (RSD) of 0.5–2.4% and between-day RSD of 2.1–4.3%.
Keywords: Dopamine;

Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma by Miranda Verschraagen; Kasper van der Born; T.H.Ursula Zwiers; Wim J.F. van der Vijgh (273-281).
Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60–600 and 87.5–700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8–102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4–104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3–10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6–7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.
Keywords: Cisplatin; Monohydrated cisplatin;

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC–tandem mass spectrometry. Whole blood samples (500 μl) were prepared by protein precipitation, followed by C18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 μg/l (r 2>0.996, n=9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n=5) at 0.5, 1.2, 20.0, and 75.0 μg/l, was 100.3–105.4% and ≤7.6%, respectively. The assay had a mean relative recovery of 94.8±3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at −80 °C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze–thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.
Keywords: Everolimus; SDZ RAD;

A sensitive plasma assay for acyclovir has been developed and validated. Acyclovir was separated from plasma components using Oasis HLB columns. Separation was obtained with no plasma interference using micellar electrokinetic chromatography (175 mM SDS) and hydroxypropyl-β-cyclodextrin (100 mM) in 90 mM borate buffer (pH 8.8) containing 0.2% NaCl. High sensitivity was achieved by large volume sample introduction and stacking. The linear range was from 20 to 10 000 ng/ml with a limit of quantitation of 20 ng/ml. This method is a viable alternative to HPLC because of its high separation and sensitivity, reproducibility, and adaptability to other nucleoside analogs.
Keywords: Acyclovir; Herpes simplex virus;

This paper presents a GC–MS confirmation method, based on large-volume programmed-temperature vaporisation (PTV) injection, for the determination of cannabinoids in plasma samples (or whole blood) with deuterium-labelled internal standards using only 25 μl of biological fluid. The analytes, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), were enriched by means of solid-phase extraction cartridges containing octadecyl-bonded silica and were, subsequently, methylated. A 20 μl aliquot of an extract in hexane was injected into a PTV in solvent split mode. Method development and the results of the analyses of standard reference material and real samples are presented and discussed. This micro-method is precise and sensitive enough to assess relevant cannabinoid levels in human blood for forensic investigations as well as for clinical applications.
Keywords: Cannabinoids;

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids by Lino Sghendo; Janet Mifsud; Roger Ellul-Micallef; Joe Portelli; Jeff S. Millership (307-315).
A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid–liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2/CYDEX-β 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and α,α-dimethyl-β-methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 μg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5–300 μg/ml concentration range for urine samples and in the 10–250 μg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.
Keywords: Ethosuximide;

A sensitive and specific liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method has been validated for the measurement of YF476 in human plasma. The method involves a simple liquid–liquid extraction procedure, chromatography of the extracts on a C18 column, atmospheric pressure chemical ionisation and detection in the multiple reaction monitoring mode. The calibration line was linear over the concentration range 0.1 ng/ml (the limit of quantification) to 25.0 ng/ml. Intra- and inter-batch precision was <14% and intra- and inter-batch accuracy was <11% over the entire calibration range. The bioanalytical method is robust and has been used for the analysis of many samples from human subjects involved in early clinical studies (Phase I).
Keywords: YF476; Benzodiazepines;

Acyclovir {9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV} is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C18 solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C8 column (150×2.1 mm, 5 μm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer–3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid–acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25–100 μg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R 2>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.
Keywords: Acyclovir;

Measurement of S-nitrosoalbumin by gas chromatography–mass spectrometry by Dimitrios Tsikas; Jörg Sandmann; Jürgen C. Frölich (335-346).
Highly contradictory data exist on the normal plasma basal levels in humans of S-nitrosoproteins, in particular of S-nitrosoalbumin (SNALB), the most abundant nitric oxide (·NO) transport form in the human circulation with a range of three orders of magnitude (i.e., 10 nM–10 μM). In previous work we reported on a GC–MS method for the quantitative determination of SNALB in human plasma. This method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, HgCl2-catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, their derivatization to the pentafluorobenzyl derivatives and quantification by GC–MS. By this method we had measured SNALB basal plasma levels of 181 nM in healthy humans. It is generally accepted that HgCl2-catalysed conversion of S-nitroso groups into nitrite is specific. In consideration of the highly divergent SNALB plasma levels in humans reported so far, we were interested in an additional method that would allow specific conversion of S-nitroso groups into nitrite. We found that treatment with cysteine plus CuSO4 is as effective and specific as treatment with HgCl2. The principle of the cysteine/CuSO4 procedure is based on the transfer of the S-nitroso group from SNALB to cysteine yielding S-nitrosocysteine, and its subsequent highly Cu2+-sensitive conversion into nitrite via intermediate ·NO formation. Similar SNALB concentrations in the plasma of 10 healthy humans were measured by GC–MS using HgCl2 (156±64 nM) and cysteine/CuSO4 (205±96 nM). Our results strongly suggest that SNALB is an endogenous constituent in human plasma and that its concentration is of the order of 150–200 nM under physiological conditions.
Keywords: S-Nitrosoalbumin; Cysteine; Copper;

A liquid chromatographic–mass spectrometric (LC–MS) method with rapid automated sample preparation was developed and validated for determination of glybenclamide in human serum. Glybenclamide and its deuterated labelled internal standard were extracted from human serum samples by automated solid-phase extraction. The extract was injected into the LC–MS system for analysis. Glybenclamide and its internal standard were measured in multiple ion monitoring mode. The method was validated over a range of 10–1000 ng/ml with good accuracy and precision and was applicable for pharmacokinetic studies.
Keywords: Glybenclamide;

Anti-low density lipoprotein antibody (anti-LDL) immobilized polyhydroxyethylmethacrylate (pHEMA) based membrane was prepared for selective removal of cholesterol from hypercholesterolemic human plasma. In order to further increase blood-compatibility, a newly synthesized comonomer, methacryloylamidophenylalanine (MAPA) was included in the membrane formulation. p(HEMA–MAPA) membranes were produced by a photopolymerization and then characterized by swelling tests, SEM and contact angle studies. Blood-compatibility tests were also investigated. The water swelling ratio of the p(HEMA–MAPA) membrane increases significantly (133.2.9%) compared with pHEMA (58%). p(HEMA–MAPA) membranes have large pores around in the range of 5–10 μm. All the clotting times increased when compared with pHEMA membranes. Loss of platelets and leukocytes was very low. The maximum anti-LDL antibody immobilization was achieved around pH 7.0. Immobilization of anti-LDL antibody was 12.6 mg/ml. There was a very low non-specific cholesterol adsorption onto the plain p(HEMA–MAPA) membranes, about 0.36 mg/ml. Anti-LDL antibody immobilized membranes adsorbed in the range of 4.5–7.2 mg cholesterol/ml from hypercholesterolemic human plasma. Up to 95% of the adsorbed LDL antibody was desorbed. The adsorption–desorption cycle was repeated 10 times using the same membrane. There was no significant loss in the adsorption capacity.
Keywords: Cholesterol;

Isolation and identification of the glucuronide of 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid from rabbit urine by Qingguang Dong; Jingkai Gu; Dafang Zhong; J. Paul Fawcett; Dafeng Chu (369-372).
The metabolic profile of 3H-1,2-dihydro-2-(4-methylphenylamino)methyl-1-pyrrolizinone (SFZ-47), a putative non-steroidal anti-inflammatory pro-drug, has been studied in rabbit urine. Semi-preparative reversed-phase HPLC of 24 h urine from two rabbits given single oral doses of SFZ-47 (200 mg) allowed the separation of SFZ-47 together with the oxidative metabolite 4-(3H-1,2-dihydro-1-pyrrolizinone-2-methylamino)benzoic acid (SFZ-47-COOH) and its glucuronide conjugate. The glucuronide was characterized by ESI-MSn and 1H NMR and shown to be the 1-O-acyl β-d-glucuronide conjugate of SFZ-47-COOH. The method gave excellent resolution of the glucuronide from endogenous constituents in urine and may be suitable for the preparation of glucuronide metabolites of other drugs.
Keywords: 4-(3H-1,2-Dihydro-1-pyrrolizinone-2-methylamino)benzoic acid glucuronide;

For toxicological purposes, a HPLC assay was developed for the simultaneous determination of risperidone and 9-hydroxyrisperidone in human plasma. After a single-step liquid–liquid extraction, both compounds were separated on a C18 column and measured at 280 nm. A good inter-assay accuracy (<116%) was achieved with inter-assay precision less than 12%. Quantification limits were 10 ng/ml. This rapid method (run time <5 min) is currently used for poison management.
Keywords: Risperidone; 9-Hydroxyrisperidone;