Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange–high-performance liquid chromatography (AE–HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2×10¹⁰ and 7×10¹¹ VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14×10⁹ VP/ml and a slope of 3.04×10⁵ VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.     

Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography–electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5–177 ng/ml (r²≥0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3–13.7 to −4.8–3.0%. The inter-day precision and accuracy ranged from 5.5–14.8 to −6.7–3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C₄ column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 mM NaClO₄ and 10 mM HClO₄. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor (k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.     

Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmacokinetic study

A method using high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE) is described for the determination of ginsenoside Rg3 in human plasma. A 2.5-ml volume of plasma was mixed with 2.5 ml 60% methanol aqueous solution, and centrifuged at 1100 g for 10 min, the supernatant fluid was further purified by SPE with 200 mg/5 ml 40 μm octadecyl silica and separation was obtained using a reversed-phase column under isocratic conditions with ultraviolet absorbance detection. The intra- and inter-day precision, determined as relative standard deviations, were less than 5.0%, and method recovery was more than 97%. The lower limit of quantitation, based on standards with acceptable RSDs, was 2.5 ng/ml. No endogenous compounds were found to interfere with analyte. A good linear relationship with a regression coefficient of 0.9999 in the range of 2.5 to 200 ng/ml was observed. This method has been demonstrated to be suitable for pharmacokinetic studies in humans. Method development for determination of drug with low UV absorption by SPE and HPLC is also discussed.     

Decay rate and substrate quality of fine roots and foliage of two tropical tree species in the Luquillo Experimental Forest,Puerto Rico

Decomposition rates, initial chemical composition, and the relationship between initial chemistry and mass loss of fine roots and foliage were determined for two woody tropical species, Prestoea montana and Dacryodes excelsa, over a gradient of sites in two watersheds in the Luquillo Experimental Forest, Puerto Rico. At all locations, fine roots decayed significantly more slowly than foliage during the initial 6 months.Substrate quality of the initial tissue showed marked differences between roots and foliage when using cell wall chemistry, secondary chemistry and total elemental analysis as indices. Quantity of acid detergent fiber (ADF) (non-digestible cell wall fiber) and lignin content were higher for roots than leaves: D. excelsa roots had 55.3% ADF and 28.7% lignin while leaves had 36.2% ADF and 11.8% lignin; P. montana roots had 68.0% ADF and 26.8% lignin while leaves had 48.5% ADF and 16.1% lignin. Aluminum concentrations were higher in fine roots (843 mg kg^–1 in D. excelsa, 1500 mg kg^–1 in P. montana) than leaves (244 mg kg^–1 in D. excelsa, 422 mg kg^–1 in P. montana), while calcium concentrations were higher in foliage (5.5 mg g^–1 in D. excelsa, 7.8 mg g^–1 in P. montana) than roots (3.4 mg g^–1 in D. excelsa, 3.1 mg g^–1 in P. montana). Nitrogen did not show any trend with tissue or species type. A linear model between mass remaining after 6 months and initial tissue chemistry could be developed only for calcium (r²=0.64).     

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).     

Analysis of cocaethylene,benzoylecgonine and cocaine in human urine by high-performance thin-layer chromatography with ultraviolet detection: a comparison with high-performance liquid chromatography

Cocaine and ethanol are frequently used at the same time, resulting in the formation of cocaethylene by transesterification. We studied the capability of high-performance thin-layer chromatography (HPTLC) to simultaneously detect cocaethylene, cocaine and benzoylecgonine in 16 urine specimens of drug addicts, previously tested as positive for benzoylecgonine at immunoenzymatic screening. Accuracy and precision, as well as detection and quantitation limits of the method, were evaluated by comparison with high-performance liquid chromatography (HPLC). HPTLC limit of quantitation was 1.0 μg/ml for the three compounds, whereas HPLC limits were 0.2 μg/ml for benzoylecgonine and cocaine, and 0.1 μg/ml for cocaethylene. The relative standard deviation (RSD) ranged from 1.03 to 12.60% and from 1.56 to 16.6% for intra- and inter-day HPTLC analysis, respectively. In the case of the HPLC method, the RSD for the intra-day precision ranged from 0.79 to 5.05%, whereas it ranged from 1.19 to 10.64% for the inter-day precision. In comparison with HPLC, HPTLC is less expensive and faster, requiring 2–3 h to analyze 10–12 samples on a single plate. In conclusion, HPTLC is suitable for determinations of the three analytes only for samples with high concentrations.     

Determination of ethyl-p-hydroxybenzoate in sow pancreatic juice by reversed-phase high-performance liquid chromatography

We have developed a high-performance liquid chromatographic–UV–Vis–diode-array detection (HPLC–DAD) method for the determination of ethyl-p-hydroxybenzoate, a hydrolytic degradation product of the synthetic protease inhibitor, gabexate-mesilate ethyl-p-(6-guanidinohexanoyloxy) benzoate methanesulfonate (GM) (FOY) in sow pancreatic juice. Methyl-p-hydroxybenzoate (I) was used as the internal standard. The pancreatic juice was deproteinised by acetonitrile and the analytes were chromatographed on a reversed-phase C₁₈ LC column using the gradient elution method. The mobile phase consisted of a solution of 0.017 M orthophosphoric acid and another solution of acetonitrile–water (80:20, v/v). The wavelength of detection was 237 nm. The limit of quantification of the method was 0.20 μM at a 9:1 signal-to-noise ratio. The overall intra- and inter-day accuracy (relative error, RE) ranged from 14.2 to 8.3% and from 13.3 to 9.8, respectively. The overall intra- and inter-day precision (relative standard deviation, RSD) ranged from 7.6 to 2.62% and from 6.7 to 3.1%, respectively. The method proved to be sensitive, specific, accurate and precise and was successfully used to determine the ethyl-p-hydroxybenzoate (II) in sow pancreatic juice.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

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.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

High-performance thin-layer chromatography method for inositol phosphate analysis

A simple and inexpensive high-performance thin-layer chromatography (HPTLC) method for the analysis of inositol mono- to hexakisphosphates on cellulose precoated plates is described. Plates were developed in 1-propanol–25% ammonia solution–water (5:4:1) and substance quantities as low as 100–200 pmol were detected by molybdate staining. Chromatographic mobilities of nucleotides and phosphorylated carbohydrates were also characterized. Charcoal treatment was employed to separate nucleotides from inositol phosphates with similar R_F values prior to HPTLC analysis. Practical application of the HPTLC system is demonstrated by analysis of grain extracts from wild type and low-phytate mutant barley as well as phytate degradation products resulting from barley phytase activity.     

Enantioselectivity of debrisoquine 4-hydroxylation in Brazilian Caucasian hypertensive patients phenotyped as extensive metabolizers

Debrisoquine (D), an antihypertensive drug metabolized to 4-hydroxydebrisoquine (4-OHD) by CYP2D6, is commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as β-adrenergic blockers, tricyclic antidepressants, and class 1C antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-OHD or S-(+) and R-(−)-4-OHD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-(−)- and S-(+)-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-OHD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-OHD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-OHD/% of dose excreted as D+4-OHD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5–acetonitrile (9:1, v/v) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-OHD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-(+)-4-OHD and R-(−)-4-OHD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5–acetonitrile–methanol (85:12:3, v/v/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter-day RSDs were less than 10% for each enantiomer. A high degree of enantioselectivity in the 4-hydroxylation of D favouring the S-(+) enantiomer was observed, resulting in R-(−)-4-OHD not detected in the urine of the EM hypertensive patients studied.     

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.     

Simultaneous determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous quantification of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma. The method involved the solid-phase extraction of the five drugs and the internal standard (I.S., verapamil) from 400 μl of human plasma. The HPLC analysis used a reversed-phase C₁₈ analytical column and a mobile phase consisting of a gradient with 15 mM phosphate buffer (pH 5.75)–acetonitrile and UV monitoring. The method was linear over the therapeutic concentration range for the five HIV-protease inhibitors. The accuracy of the method ranged from 98.2 to 106.7% and the precision values ranged from 1.4 to 8.1% for intra-day precision and from 3.1 to 6.4% for the inter-day values.     

Specific high-performance liquid chromatography assay for determination of rifabutin plasma concentration following Extrelut column extraction

A specific, precise and accurate assay for determination of rifabutin in human plasma using Extrelut column extraction was developed and validated. Rifabutin concentrations were calculated with a standard curve ranging from 5 to 800 ng ml⁻¹, using a split-curve approach. Chromatographic peaks were separated by means of a 5 μm Symmetry Shield RP8 using a KH₂PO₄ (0.05 M) buffer–acetonitrile mobile phase. Detection wavelength was set at 275 nm. Chromatography was carried out at room temperature (20–25°C). The limit of quantification was 5 ng ml⁻¹. The recovery was over 71%. The intra-day precision of the assay was 5, 7, and 1% while the inter-day precision was 11.2, 8.1, and 5.8% at concentrations of 30, 150 and 500 ng ml⁻¹, respectively. The accuracy ranged from 99 to 108%. Forty of the drugs most commonly administered to HIV-positive patients were found not to interfere with the assay. The assay has been used in a comparative study of rifabutin pharmacokinetics in HIV-positive patients with or without wasting syndrome.     

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.