High-performance liquid chromatographic method for the determination of cefpodoxime levels in plasma and sinus mucosa

A selective HPLC method is described for the determination of cefpodoxime levels in plasma and sinus mucosa. Sample preparation included solid-phase extraction with a C₈ cartridge. Cefpodoxime and cefaclor (internal standard) were eluted with methanol and analyzed on an optimised system consisting of a C₁₈ stationary phase and a ternary mobile phase (0.05 M acetate buffer pH 3.8—methanol—acetonitrile, 87:10:3, v/v) monitored at 235 nm. Linearity and both between- and within-day reproducibility were assessed for plasma and sinus mucosa samples. Inter-assay coefficients of variation were lower than 13.6% (n = 10) for plasma (0.2 μg/ml) and lower than 12.4% (n = 5) for sinus mucosa (0.25 μg/g). The quantification limit was 0.05 μg/ml for plasma and 0.13 μg/g for tissue. The method was used to study the diffusion of cefpodoxime in sinus mucosa.     

Determination of the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C₁₈, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C₈ cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20–1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.     

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.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Small blood volumes from children for quantitative sotalol determination using high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method using fluorescence detection has been developed for sotalol determination in small plasma samples of children and newborns with limited blood volume. In sample sizes of 100 μl of plasma, sotalol was extracted using an internal standard and solid-phase extraction columns. Chromatographic separation was performed on a Spherisorb C₆ column of 150×4.6 mm I.D. and 5 μm particle size at ambient temperature. The mobile phase consisted of acetonitrile–15 mM potassium phosphate buffer (pH 3.0) (70:30, v/v). The excitation wavelength was set at 235 nm, emission at 300 nm. The flow-rate was 1 ml/min. Sotalol and the internal standard atenolol showed recoveries of 107±8.9 and 97±8.1%, respectively. The linearity range for sotalol was between 0.07 and 5.75 μg/ml, the limit of quantitation 0.09 μg/ml. Precision values expressed as percent relative standard deviation of intra-assay varied between 0.6 and 13.6%, that of inter-assay between 2.4 and 14.4%. Accuracy varied between 86.1 and 109.8% (intra-assay) and 95.4 and 103.3% (inter-assay). Other clinically used antiarrhythmic drugs did not interfere. As an application of the assay, sotalol plasma concentrations in a 6-year-old child with supraventricular tachycardia treated with oral sotalol (3.2 mg/kg per day) are reported.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Determination of gabapentin in plasma by high-performance liquid chromatography

A rapid and simple method for determination of the novel antiepileptic compound gabapentin [1-(aminomethyl)cyclohexaneacetic acid] in plasma is described. Blank human plasma was spiked with gabapentin (1.0–10.0 μg/ml) and internal standard [1-(aminomethyl)-cycloheptaneacetic acid; 5.0 μg/ml]. Individual samples were treated with 2 M perchloric acid, centrifuged and then derivatised with o-phthalaldehyde-3-mercaptopropionic acid. Separation was achieved on a Beckman Ultrasphere 5 μm reversed-phase column with mobile phase consisting of 0.33 M acetate buffer (pH 3.7; containing 100 mg/l EDTA)-methanol-acetonitrile (40:30:30, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 330 and 440 nm, respectively. The calibration curve for gabapentin in plasma was linear (r=0.9997) over the concentration range 1.0–10.0 μg/ml. Recovery was seen to be 90%. The inter- and intra-assay variations for three different gabapentin concentrations were 10% throughout. The lower limit of quantitation was found to be 0.5 μg/ml. Chromatography was unaffacted by a range of commonly employed antiepileptic drugs or selected amino acids.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C₁₈ column (250 × 4.6 mm I.D.) using 10 mM phosphoric acid—acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 μg/ml and the detection limits were 0.06 μg/ml for indomethacin and 0.08 μg/ml for mefenamic acid, for 50μl plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Measurement of plasma uracil using gas chromatography–mass spectrometry in normal individuals and in patients receiving inhibitors of dihydropyrimidine dehydrogenase

A sensitive gas chromatographic–mass spectrometric method is described for reliably measuring endogenous uracil in 100 μl of human plasma. Validation of this assay over a wide concentration range, 0.025 μM to 250 μM (0.0028 μg/ml to 28 μg/ml), allowed for the determination of plasma uracil in patients treated with agents such as eniluracil, an inhibitor of the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase. Calibration standards were prepared in human plasma using the stable isotope, [¹⁵N₂]uracil, to avoid interference from endogenous uracil and 10 μM 5-chlorouracil was added as the internal standard.     

Simultaneous high-performance liquid chromatographic assay of droperidol and flunitrazepam in human plasma: Application to haemodilution blood samples collected during clinical anaesthesia

A simultaneous assay for droperidol and flunitrazepam by high-performance liquid chromatography has been developed and applied to blood samples collected during an acute normovolemic haemodilution under general anaesthesia. Haemodilution blood samples were stored at +4°C to be transfused, if required, to a patient during the post-surgical phase. A C₁₈ Supelclean cartridge was used for solid-phase extraction, and the recoveries were 74% and 89%, respectively, for droperidol and flunitrazepam. Compounds were chromatographed on a C₁₈ Novapak column at 250 nm, with a mobile phase of acetonitrile—10 mM ammonium acetate buffer (pH 6.7) (45:55, v/v). Nitrazepam was used as the internal standard. For both drugs, the assay was linear up to 500 μg/l, and the detection limits were 20 and 10 μg/l for droperidol and flunitrazepam, respectively, and their observed levels in haemodilution samples were 93 ± 82 μg/l and 76 ± 107 μg/l, respectively. Some of the values for flunitrazepam were higher than the minimal efficient concentration, defined as the plasma level observed at the time of the patient wakening from anaesthesia (12 ± 4 μg/l). According to our results, haemodilution sampling can be performed before induction of anaesthesia. When the blood is collected after the anaesthetic induction, it seems necessary to determine levels of the two drugs in haemodilution samples to avoid side-effects.     

Rapid and simple high-performance liquid chromatographic determination of nimesulide in human plasma

A high-performance liquid chromatographic method for the quantitation of nimesulide in human plasma is presented. The method is based on protein precipitation with methanol and reversed-phase chromatography with spectrophotometric detection at 404 nm. The separation was performed on a Nucleosil 120-5 C₁₈, 50×4-mm I.D. column and the mobile phase consisted of acetonitrile–methanol–15 mM potassium dihydrogenphosphate buffer, pH 7.3 (30:5:65, v/v). Only 250 μl of plasma are used for sample preparation and no internal standard is necessary. The limit of quantitation is 80 ng/ml and the calibration curve is linear up to 10 000 ng/ml. More than 20 samples can be analysed within 1 h. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.