Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Validated high-performance liquid chromatographic method utilizing solid-phase extraction for the simultaneous determination of naringenin and hesperetin in human plasma

Naringenin and hesperetin, the aglycones of the flavanone glucosides naringin and hesperidin occur naturally in citrus fruits. They exert a variety of pharmacological effects such as antioxidant, blood lipid-lowering, anticarcinogenic and inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A specific, sensitive, precise, and accurate solid-phase extraction high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of naringenin and hesperetin in human plasma was developed and validated. After addition of 7-ethoxycoumarin as internal standard, plasma samples were incubated with beta-glucuronidase/sulphatase, and the analytes were isolated from plasma by solid-phase extraction using C(18) cartridges and separated on a C(8) reversed phase column with methanol/water/acetic acid (40:58:2, v/v/v) as the eluent at 45 degrees C. The method was linear in the 10-300 ng/ml concentration range for both naringenin and hesperetin (r>0.999). Recovery for naringenin, hesperetin and internal standard was greater than 76.7%. Intra- and inter-day precision for naringenin ranged from 1.4 to 4.2% and from 1.9 to 5.2%, respectively, and for hesperetin ranged from 1.3 to 4.1% and from 1.7 to 5.1%, respectively. Accuracy was better than 91.5 and 91.3% for naringenin and hesperetin, respectively.     

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).     

Rapid and selective high-performance liquid chromatographic method for the determination of metronidazole and its active metabolite in human plasma, saliva and gastric juice

A rapid and selective HPLC method has been developed for the separation and quantitation of metronidazole and its hydroxylated metabolite in human plasma, saliva and gastric juice. The assay requires a simple protein precipitation step prior to analysis and is selective, sensitive and reproducible. The limits of quantitation (0/5-ml sample) were at least 0.25 μg/ml for metronidazole and 0.20 μg/ml for its hydroxy metabolite. A Hypersil ODS 5 μm (150×4.6 mm I.D.) column was used with a mobile phase of acetonitrile-aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90) delivered at a flow-rate of 1.0 ml/min.     

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.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 μm) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5–100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (−)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C₁ stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (−)-nicardipine was possible in a concentration range of 5–100 ng ml⁻¹ and the limit of detection in plasma was 2.5 ng ml⁻¹. The recoveries of (+)- and (−)-nicardipine added to plasma were 91.4–98.4% and 93.3–96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (−)-nicardipine in plasma from healthy volunteers.     

Rapid high-performance liquid chromatographic determination of vancomycin in human plasma

A rapid simple and robust reversed-phase HPLC method was developed for rapid screening in bioavailability studies or comparative bioequivalence studies. The method is specific for vancomycin as no interference from acetylsalicylic acid, paracetamol and caffeine was observed. The mean intra-day precision was from 11.7% (low concentration) to 0.3% (high concentration) and the within-day precision from 15.0 to 0.3%, determined on spiked samples. The accuracy of the method was 106.4–99.8% (intra-day) and 103.5–100.2% (inter-day).     

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.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.