Liquid chromatographic analysis of the cis(Z)- and trans(E)-isomers of clopenthixol in human plasma using a novel solid phase extraction procedure

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in human plasma has been developed. The chromatographic analysis was carried out isocratically on a reversed-phase column (C(8) 150 x 4.6 mm I.D., 5 microm) using a mixture of 25 mM phosphate buffer and acetonitrile (65:35 v/v, pH* 3.0) as the mobile phase, and ultraviolet detection at 230 nm. Plasma sample pretreatment was accomplished by means of an original solid-phase extraction (SPE) procedure carried out on cyanopropyl cartridges, with a high extraction yield and good selectivity. Under the optimum conditions, calibration graphs of spiked human plasma samples were obtained over the concentration ranges 1-300 ng ml(-1) for cis(Z)-clopenthixol and 1-200 ng ml(-1) for trans(E)-clopenthixol. The limit of detection (LOD) was 0.3 ng ml(-1) for both cis(Z)- and trans(E)-isomers of clopenthixol. The method was successfully applied to the determination of cis(Z)-clopenthixol and trans(E)-clopenthixol in plasma samples of schizophrenic patients undergoing therapy with zuclopenthixol.     

Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum

A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile—phosphoric acid (85%)—water (20:2:78, v/v/v), followed by ultrafiltration through a 10 000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol—acetonitrile—phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable photometric UV-Vis detector set at 215 nm, and confirmed by a photodiode array detector operated in the 200–320 nm range. The limit of detection for ketamine was 5–15 ng/ml in equine serum. Additionally, the dehydronorketamine peak identity was tentatively confirmed by thermospray liquid chromatography—mass spectrometry.     

Synthesis and antiherpetic activity of (Z)- and (E)-isomers of 9-(3-phosphonomethoxyprop-1-en-yl)adenine

9-(3-Phosphonomethoxyprop-1-en-yl)adenine (Z)- and (E)-isomers were synthesized. The stereoselectivity of double bond formation was studied by variation of sulfonyl groups. The resulting phosphonates exhibited a moderate antiherpetic activity in a culture of Vero cells infected with herpes simplex type 1 virus. The Z-isomer was shown to be more effective inhibitor of virus reproduction in the case of both wild and acyclovir-resistant strain.     

Development of a high-performance liquid chromatographic method for the analysis of staurosporine

Staurosporine (Stsp), a protein kinase inhibitor, has been found to have a differential effect on the proliferation of normal and transformed cells in vitro. Hence, Stsp might be used in cancer therapy to arrest normal proliferating cells in G1, while permitting tumor cells to continue proliferation. The patient could then be treated with a therapeutic agent of maximum toxicity for actively proliferating tumor cells. To facilitate investigations of Stsp in vivo, we have developed an HPLC method for measuring the levels of Stsp in blood. Using a rat model, plasma containing Stsp is treated with acetone to precipitate proteins and extract the Stsp. The acetone extract is then subjected to reversed-phase HPLC on a μBondapak C₁₈ column. Using a linear elution gradient of acetonitrile containing trifluoroacetic acid, Stsp elutes as a sharp peak at ca. 35 min which can be detected by UV absorption at 292 nm. No blood or reagent components interfere with its quantification. The calibration curve, ranging from 0.1 to 2.0 μg Stsp, demonstrated a linear response to Stsp concentration having a correlation coefficient (r²) of 0.990. Precision analysis demonstrated that the method will yield results that are ±11.6% from the mean 95% (two standard deviations) of the time. This method was used to measure Stsp levels in plasma after administering an injection of 0.2 mg Stsp into the jugular vein of rats. No Stsp could be detected in the plasma 5 min after injection, even though enough Stsp was administered to be easily detectable if it was simply contained in the plasma. Thus, it is concluded that some compartment other than the plasma must adsorb the Stsp from the plasma and sequester it in vivo.     

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125×4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 μl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 μg/ml and was capable of detecting a minimum concentration of 0.029 μg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Specific high-performance liquid chromatographic assay with ultraviolet detection for the determination of 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea in plasma

A facile, sensitive and highly specific HPLC method for assaying 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in plasma has been developed. The drug was efficiently isolated from plasma by extraction with tert.-butyl methyl ether. A structurally related compound with similar physicochemical properties served as the internal standard (I.S.). Following evaporation of the organic solvent, the extract was reconstituted with 0.05 M ammonium acetate buffer, pH 5.0, and loaded onto a 4 μm Nova-Pak C₁₈ column (15 cm×3.9 mm), which was preceded by a 7 μm Brownlee RP-18 precolumn (1.5 cm×3.2 mm). Chromatography was performed at ambient temperature using a mobile phase of methanol-0.1 M ammonium formate buffer, pH 3.7 (25:75, v/v). UV absorbance of the effluent was monitored at 240 nm. A flow-rate of 1.0 ml/min was used for analyzing mouse and dog plasma extracts. Under these conditions, the drug eluted at 4.0 min and was followed by the I.S. at 6.1 min. An automatic switching valve was employed to allow the precolumn to be flushed 1.5 min into the run, without interrupting the flow of the mobile phase to the analytical column, thereby preventing the apparent build-up of extractable, strongly retained, UV-absorbing components present in mouse and dog plasma. Operating in this manner, more than 100 samples could be analyzed during a day using a refrigerated autosampler for overnight injection. The method was readily adapted to the determination of SarCNU in human plasma by simply decreasing the eluent flow-rate to 0.6 ml/min, whereby SarCNU and the I.S. eluted at approximately 5.8 and 9.1 min, respectively. Furthermore, the switching valve was not necessary for the analysis of human plasma samples. With a 50-μl sample volume, the lowest concentration of SarCNU included in the plasma standard curves, 0.10 μg/ml, was quantified with a 7.8% R.S.D. (n=27) over a 2 month period. Plasma standards, with concentrations of 0.26 to 5.1 μg/ml, exhibited R.S.D. values ranging from 1.3 to 4.7%. Thermospray-ionization MS detection was used to definitively establish the specificity of the method. The sensitivity of the assay was shown by application to be more than adequate for characterizing the plasma pharmacokinetics of SarCNU in mice.     

Sensitive gas-liquid chromatographic method for the assay of the neuroleptic drug cis(Z)-flupentixol in human serum or plasma

A gas-liquid chromatographic (GLC) assay suitable for the analysis of the cis(Z)-stereoisomer of the antipsychotic drug flupentixol in human serum or plasma was developed. The minimal quantifiable concentration was 0.5 ng/ml and the day-to-day coefficient of variation was 11.2% at 1 ng/ml and 8.7% at 10 ng/ml. Following addition of perphenazine as the internal standard (I.S.) and aqueous NaOH, samples (2 ml) are extracted with n-hexane-isoamyl alcohol (98.5:1.5, v/v) (solvent), back-extracted to 0.1 M HCl and after one washing-step and addition of aqueous NaOH again extracted into 100 μl solvent. After evaporation to dryness, the extract is reconstituted in 20 μl solvent and evaporated to approximative 10 μl. A 4-μl aliquot is injected cool on-column onto the GLC system. A gas chromatograph HP 5890 with on-column injection port, nitrogen-phosphorus detector (NPD), a HP-1 25 m × 0.32 mm I.D., 0.5 μm capillary and hydrogen (3 ml/min, automated pressure control) as the carrier gas was applied. The negative influence of light on the assay was measured and discussed. The suitability of this method for clinical pharmacokinetic studies and therapeutic drug monitoring (TDM) was determined by the analysis of serum samples of 12 schizophrenic patients.     

Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride

An improved HPLC method using a silica gel column with fluorescence detection (excitation at 300 nm and emission at 365 nm) was developed for the determination of sulpiride concentrations in plasma. Analysis of sulpiride in plasma samples was simplified by a one-step liquid–liquid extraction after alkaline treatment of only 1 ml of plasma. The low limit of quantitation was 20 ng/ml with a coefficient of variation of less than 20%. A linear range was found from 20 to 1500 ng/ml. This HPLC method was validated with the precision for inter-day and intra-day runs being 0.36–8.01% and 0.29–5.25%, respectively, and the accuracy (standard deviation of mean, SD) for inter-day and intra-day runs being −1.58 to 5.02% and −2.14 to 5.21%, respectively. Bioequivalence of the two products was evaluated in 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment cross-over study. Sulpiride plasma concentrations were analyzed with this validated HPLC method. Results demonstrated that the two tablet formulations of sulpiride appear to be bioequivalent.     

Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-¹⁴C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.     

Subtraction method for the high-performance liquid chromatographic measurement of plasma adenosine

The measurement of plasma adenosine with traditional high-performance liquid chromatographic techniques is difficult because of its nanomolar concentration, its short half-life in blood, and because of the difficulty in isolating adenosine from interfering peaks in the chromatogram. To prevent loss of adenosine in the blood sample, a “stop solution” is used to prevent enzymatic degradation and cellular uptake. Peak-shifting techniques on fractionated samples to measure adenosine derivatives have been used in the past to avoid interfering peaks in the chromatogram. A new method has been developed by which nanomolar levels of plasma adenosine can be accurately measured despite co-eluting peaks in the chromatogram. In this method, plasma samples are collected with a stop solution, processed, and divided. Adenosine deaminase is added to part of the sample to form a blank. A computer program subtracts the blank chromatogram from the paired unknown, and the result is compared to adenosine standards prepared from the blank and subtracted in a similar fashion. With this subtraction method, the overall recovery of physiological concentrations of adenosine was 89% from dog blood, and the average coefficient of variation was 12%. In summary, the subtraction method of plasma adenosine measurement offers good recovery, reproducibility, and the ability to quantify low levels of adenosine despite interfering peaks in the chromatogram.     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithio-carbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 μl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150×3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 μg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 μg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 μg/ml in plasma (500 μl) using a weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ration values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 μg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 μl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Sensitive high-performance liquid chromatographic method for the determination of coumarin in plasma

A high-performance liquid chromatographic method was developed for the determination of coumarin in plasma at low concentrations. The method involves a single-step extraction of the alkalinized sample with hexane and subsequent evaporation of the organic phase in the presence of hydrochloric acid to collect and concentrate the coumarin. Analysis of the acidic phase was performed on a C₈ column and coumarin was detected by measuring the UV absorbance at 275 nm. The limit of detection was 0.3 μg l⁻¹. The assay was used to study the evolution of concentrations of coumarin in one volunteer after oral administration of a single 10-mg dose.     

Simple and rapid high-performance liquid chromatographic method for the quantification of 3-methylhistidine

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C₁₈ analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [¹⁴C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

A high-performance liquid chromatographic method for the determination of polyphosphoinositides in brain

A simple high-performance liquid chromatographic method for the determination of endogenous phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in brain has been developed. PIP and PIP2 were derivatized with 9-anthryldiazomethane to yield (9-anthryl)PIP and di(9-anthryl)-PIP2. The derivatives were separated on a reversed-phase column using isocratic elution and detected with a uv detector. The detection limits of PIP and PIP2 were 0.25 micrograms. The method with uv detection was sufficiently sensitive to measure the concentrations of PIP and PIP2 in rat brain. The levels of PIP and PIP2 were increased in developing rat brain and were decreased after 10 min of ischemia.     

Sensitive high-performance liquid chromatographic method for a dopamine receptor agonist,CI-1007, and its metabolite PD 147693 in monkey plasma

A sensitive gradient high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of a dopamine autoreceptor agonist CI-1007 (I) and its metabolite PD 147693 (II) is described. Monkey plasma samples were purified by liquid-liquid extraction using hexane. Liquid chromatographic separation was achieved on two C₁₈ analytical columns (installed in series) using gradient elution. Column effluent was monitored using a fluorescence detector programmed to change wavelengths at specified times. Minimum quantitation limits of I and II were 3.0 and 5.0 ng/ml, respectively, for a plasma sample volume of 0.100 ml. Linearity was demonstrated up to 300 ng/ml. The assay has been applied to the analysis of I and II in plasma from monkeys following intravenous and oral doses of I.