Development and validation of a sensitive solid-phase extraction and high-performance liquid chromatography assay for the novel antitumour agent CT2584 in human plasma

A HPLC assay and solid-phase extraction technique from human plasma has been developed and validated for the novel anticancer agent CT2584, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, which has recently completed a phase I trial at the Christie Hospital, Manchester under the auspices of the CRC phase I/II committee. Following addition of CT2576, 1-(11-octylamino-10-hydroxylundecyl)-3,7-dimethylxanthine, as internal standard, a solid-phase extraction cartridge (100 mg cyanopropyl) was used to isolate the drug CT2584 from human plasma. Analysis was performed by reversed-phase chromatography. CT2576 was used as internal standard at a concentration of 4 μg ml⁻¹ for the quantification of CT2584 from plasma for the duration of this work. The lower limit of quantification for the drug CT2584 in buffer using this assay was found to be 0.0122 μM (0.008 μg ml⁻¹) and 0.048 μM (0.027 μg ml⁻¹) when extracted from human plasma.     

Development and validation of a high-performance liquid chromatographic assay using solid-phase extraction for the novel antitumor agent pancratistatin in human plasma

The stability of the experimental anti-tumour agent pancratistatin in human plasma has been investigated. A solid-phase extraction technique and an HPLC assay with external standards have been developed and validated. Extraction was performed using C₁₈ cartridges and HPLC, analysis was performed on a 15 cm Hypersil BDS column using isocratic elution with 13% acetonitrile and aqueous solution of 1% (w/v) acetic acid. The lower limit of quantification for pancratistatin in 5% DMF–95% water was found to be 0.58 ng/ml (±10.58%) and 2.3 ng/ml (±9.2%) following extraction from human plasma. Mean recovery of 89.4% (±4.73%) was obtained over the concentration range 0.0023–9.45 μg/ml for a five day validation study. Pancratistatin was stable at room temperature in light or dark for at least 15 days, in the refrigerator at 4°C for at least 16 days and in the freezer at −20°C or −80°C for at least 28 days. Under all conditions monitored, % recovery of pancratistatin from human plasma was greater than 95% and no evidence of degradation had occurred. There also was no loss of pancratistatin after three cycles of freezing and thawing.     

Determination of alentamol hydrobromide, a novel antipsychotic agent, in human blood plasma and urine by high-performance liquid chromatography with fluorescence detection and solid-phase extraction

Alentamol hydrobromide, (+)-2-(dipropylamino)-2,3-dihydro-1H-phenalen-5-ol monohydrobromide, is a selective dopamine agonist currently being investigated for the treatment of schizophrenia. This paper describes a reversed-phase high-performance liquid chromatographic-based method for the quantification of alentamol in blood plasma and urine. The method utilizes solid-phase extraction with carboxylic acid-derivatized silica columns. A limit of quantitation of 0.1 ng/ml in plasma was achieved by virtue of selective extraction and fluorescence detection. Example chromatograms of plasma and urine specimens from clinical trials demonstrate the utility of the method.     

Simple and sensitive assay of zonisamide in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A rapid and sensitive method for the assay of zonisamide in serum was developed using a solid-phase extraction technique followed by high-performance liquid chromatography. A 20-μl volume of human serum was first purified with a Bond-Elut cartridge column. Then, the methanol eluate was injected onto a reversed-phase HPLC column with a UV detector. The mobile phase was acetonitrile—methanol—distilled water (17:20:63, v/v) and the detection wavelength was 246 nm. The detection limit was 0.1 μg/ml in serum. The coefficients of variation were 4.2–5.6% and 5.1–9.1% for the within-day and between-day assays, respectively. This method can be used for clinical pharmacokinetic studies of zonisamide in serum even in infant patients with epilepsy.     

Development of an assay for histamine using automated high-performance liquid chromatography with electrochemical detection

Previous studies have measured histamine by derivatization with o-phthaldialdehyde (OPA) and mercaptoethanol (ME), followed by reversed-phase HPLC separation and electrochemical detection. The derivatization product, however, was very unstable. In the present study, inclusion of less polar solvents (e.g., acetonitrile or tetrahydrofuran) in the OPA/ME derivatization reaction produced an OPA/ME-histamine product that was stable for many hours. Changes of the HPLC mobile phase (increasing its ionic strength and pH and including triethylamine) dramatically improved the chromatography and reduced the histamine detection limit to <0.1 pmol. The modified assay was suitable for batchwise manual derivatization of histamine samples followed by their automated analysis by HPLC with an automatic injector.     

Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography

We developed a sensitive assay to measure amoxicillin in human plasma and midle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50–200 μl of sample with Bond Elut C₁₈ cartridges. The exact was analyzed on a 15 cm × 2 mm, 5μm Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 ± 16.6% and 91.0 ± 6.8%, respectively. The limit of quantitation was 0.125 μg/ml with 200 μl sample size. The linear range was from 0.125 to 35.0 μg/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.     

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300×3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (λ_ex 230 nm, λ_em 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 μg/ml with a detection limit of 0.2 μg/ml. This assay is suitable for use in clinical studies with etoposide.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 μg/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C₁₈/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125×4 mm C₁₈ column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between −5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in biovailability studies for the development of a new oral penicillin-V formulation.     

Purification of fatty acid ethyl esters by solid-phase extraction and high-performance liquid chromatography

We have developed a two-step method to purify fatty acid ethyl esters (FAEE) using solid-phase extraction (SPE), with a recovery of 70±3% (mean±S.E.M.) as assessed using ethyl oleate as a recovery marker from a standard lipid mixture in hexane. The first step of the SPE procedure involves application of a lipid mixture to an aminopropyl-silica column with simultaneous elution of FAEE and cholesteryl esters from the column with hexane. Gas chromatographic analysis of FAEE without interference from cholesteryl esters may be performed using the eluate from the aminopropyl-silica column, thus eliminating the need for an octadecylsily (ODS) column in this case. The FAEE can then be separated from the cholesteryl esters, if necessary, by chromatography on an ODS column and elution with isopropanol-water (5:1, v/v). Both the aminopropyl-silica and ODS columns were found to be effective for up to four uses. To permit isolation of specific FAEE species following isolation of total FAEE by the two-step SPE method, we have also developed a purification scheme for individaal FAEE by high-performance liquid chromatography (HPLC). Thus, this simple method allows for reproducible isolation of total FAEE by SPE and isolation of individual FAEE species by HPLC.     

Determination of methylnaltrexone in clinical samples by solid-phase extraction and high-performance liquid chromatography for a pharmacokinetics study

A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 microm, 150x3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n = 10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C(max) = 110 (+/-55) and 287 (+/-101) ng/ml and t(max) = 16.7 (+/-10.8) and 20.0 (+/-9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.     

Development of a novel method for triterpenoidal saponins in rat plasma by solid-phase extraction and high-performance liquid chromatography tandem mass spectrometry

A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3, and 23-hydroxybetulinic acid) in rat plasma following solid-phase extraction (SPE). The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multiswitching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18 column (250 × 4.6 mm, 5 μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as follows: m/z 1219.7/749.4 for anemoside B4, m/z 819.4/347.2 for pulsatilloside B, m/z 749.6/471.2 for anemoside A3, m/z 471.4/471.4 for 23-hydroxybetulinic acid, and m/z 461.1/285.0 for the internal standard. The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.     

Determination of nortriptyline in human serum by fully automated solid-phase extraction and on-line high-performance liquid chromatography in the presence of antipsychotic drugs

A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C₁₈ column with a mobile phase of acetonitrile–0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid–liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.     

Determination of metronidazole in vaginal tissue by high-performance liquid chromatography using solid-phase extraction

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of metronidazole in vaginal tissue is reported. The method uses a Zorbax SB phenyl column with a 0.01 M aqueous monobasic potassium phosphate buffer (pH 4.0)-absolute methanol (85:15, v/v) as mobile phase at a flow-rate of 1.0 ml/min and detection at 313 nm. Tinidazole was used as the internal standard. The method employed homogenization of tissue followed by solid-phase extraction. The quantitation was achieved within 30 min with sensitivity in the ng/g range. Metronidazole was linear in the 100–2000 ng/g range. The accuracy and precision were in the 1–4% range for the drug and the limit of detection was approximately 100 ng/g based on a signal-to-noise ratio of 3 and a 100-μl injection.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Determination of hyperforin in human plasma using solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.     

Quantitative analysis of urinary daidzein and equol by gas chromatography after solid-phase extraction and high-performance liquid chromatography

Daidzein and its main metabolite equol are isoflavone phytoestrogens. Several studies have suggested that intake of an isoflavone-rich diet may prevent hormone-related cancer and estrogen-related disorders (cardiovascular disease, osteoporosis and menopausal symptoms). To better understand the role of isoflavones in preventing such severe disease, several methods have been developed to measure these compounds in biological fluids. However, the analytical procedures to measure isoflavones are often time-consuming and require highly skilled technicians. In this paper we describe a method for urinary daidzein and equol measurement that combines solid phase extraction and HPLC purification before gas chromatographic determination. The specificity of the method was confirmed by the gas chromatography-mass spectrometry technique. The mean recovery of daidzein and equol was 94.6% and 97.0%, respectively. The repeatability of the method was in the range of 2.0-7.4% for daidzein and 1.3-4.9% for equol. A linear relationship between observed and expected values was found in the dilution (r2=0.9983 for daidzein; r2=0.9982 for equol) and addition (r2=0.9984 for daidzein; r2=0.9989 for equol) assays. The method is suitable to measure changes in the urinary excretion of isoflavones and to investigate urinary isoflavonoids as biomarkers of isoflavone exposure.     

Assay for levormeloxifene, a selective estrogen receptor modulator, in human and monkey plasma employing high-performance liquid chromatography and solid-phase extraction

Assays for levormeloxifene, a new selective estrogen receptor modulator, and its 7-desmethyl metabolite in human and cynomolgus monkey plasma are described. Plasma was extracted on mixed-mode bonded sorbent material (C₈/SCX) and the extracts were analysed by high-performance liquid chromatography with fluorescence detection. Recoveries of levormeloxifene and the metabolite exceeded 70%. Within and total assay precision calculated as a coefficient of variation (C.V.) were <8% for both compounds at all concentration levels, except at the limit of quantitation (LOQ) where the C.V. was 15%. Within and total-assay accuracy calculated as a percentage of the nominal value were between 90 and 114% for both analytes. The LOQ was for levormeloxifene and 7-desmethyllevormeloxifene, respectively, 1.5 and 2.5 ng/ml (man) and 5.2 and 6.9 ng/ml (monkey). In the monkey plasma assay, human plasma could substitute monkey plasma as blank plasma.     

Determination of ketamine and norketamine enantiomers in plasma by solid-phase extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method is described for determination of sub-anaesthetic concentrations of the enantiomers of ketamine and its metabolite norketamine in plasma. The samples are purified by reversed-phase solid-phase extraction. The enantiomers are separated on a Chiral AGP column with a mobile phase containing 16% methanol and a 10 mM phosphate buffer at pH 7.0, and measured by UV-detection at a wavelength of 220 nm. Linear calibration curves with correlation coefficients better than 0.995 have been obtained in the range 10–320 ng/ml. Minimum detectable concentrations were about 2 ng/ml.