High-performance liquid chromatography quantiation of n-acetylneuraminic acid in malignant melanoma and breast carcinoma

A rapid isocaratic high-performance liquid chromatography (HPLC) method for quantitation of serum N-acetylneuraminic acid (NANA) is described. Separation is achieved on an Aminex HPX-87 cation-exchange resin using a 0.006 N sulfuric acid mobile phase. Compared with the more conventional thiobarbituric acid (TBA) method, HPLC is more reliable and has a much improved maximum sensitivity of 0.8 nmol/ml. In a limited study of malignant melanoma patients' sera, HPLC gave slightly higher values for NANA than TBA. In a more detailed study of breast carcinoma patients with measurable tumour burden, HPLC and TBA methods were used on the same sera and compared with concurrent carcinoembryonic antigen (CEA) determinations. HPLC resulted in a clear improvement in discrimination between tumour burden groups compared with either the TBA method or CEA.     

Analysis of pipecolic acid in biological fluids using capillary gas chromatography with electron-capture detection and [H11]pipecolic acid as internal standard

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using capillary gas chromatography with electron-capture detection. The method utilizes [²H₁₁]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction at pH ≤ 2 and further derivatization of the carboxyl moiety with pentafluorobenzyl bromide, the excess of which was removed by solid-phase extraction. Control values have been determined in the plasma of at-term infants, age > 1 week (n = 21, mean = 1.36 μM, range = 0.47–3.27 μM). The utility of the method was demonstrated by quantitating pipecolic acid in biological fluids derived from patients with peroxisomal disorders. The method was validated against an established electron-capture negative ion mass fragmentographic technique.     

Sensitive determination of docetaxel in human plasma by liquid–liquid extraction and reversed-phase high-performance liquid chromatography

A sensitive reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantitative determination of docetaxel (I) in human plasma. The concentrations in plasma, for validation procedures spiked with known amounts of I, are read from calibration curves in the range of 10–20 000 ng/ml. The sample preparation involved a liquid–liquid extraction of 1000 μl of sample with a mixture of acetonitrile–n-butylchloride (1:4, v/v). The related compound paclitaxel (II) was used as internal standard. Chromatographic separations were performed an Inertsil ODS-80A column, with UV detection performed at 230 nm. The overall extraction recoveries were 84.3 and 90.0% for I and II, respectively. The lower limit of quantitation was 10 ng/ml, and the accuracy, within-run and between-run precisions at three tested concentrations fell within the generally accepted criteria for bioanalytical assays.     

Sialic acid and the surface charge associated with hyperpolarization-activated,inward rectifying channels

Summary The whole-cell configuration of the patch-clamp technique was used with cultured pacemaker cells from the rabbit sinoatrial node to test the hypothesis that sialic acid residues (NANA) constitute much of the negative surface charge associated with hyperpolarization-activated, inward rectifying channels. Activation-voltage relationships (between –70 and –140 mV) were determined for hyperpolarization-activated (inward rectifying) current (i _f). Addition of 10mm Ca²⁺ shifted the half-activation potential (V _1/2) from –89.5±0.9 mV to –77.9±2.6 mV (P<0.01), confirming the presence of negative fixed charges on the myocytes after 3 to 5 days in culture. Addition of 20mm dimethonium, an organic divalent cation that screens but does not bind to negative surface charge, shiftedV _1/2 from –86.8±1.4 mV to –75.0±1.7 mV (P<0.001) without affecting the amplitude of the current. In contrast, 10mm Ca²⁺ reduced the amplitude ofi _f significantly. Incubation of cells with a highly purified preparation of neuraminidase (0.1–2.0 U/ml, 1 hr, 37°C), an enzyme that selectively removes NANA from glycoproteins and glycolipids, failed to alterV _1/2 or the amplitude ofi _f significantly. Pretreatment of cells with neuraminidase (1.0 U/ml, 1 hr, 37°C) failed to alter the positive shift ofV _1/2 produced by dimethonium. The results suggest that NANA does not constitute the negative surface charge associated with hyperpolarization-activated, inward rectifying channels.     

The use of lectins in the quantitation and analysis of macromolecules by affinoelectrophoresis

The technique of rocket affinoelectrophoresis, initially introduced for the quantitation of a succinylated mannan by concanavalin A [Owen, P., and Salton, M. R. J. (1976) Anal. Biochem. 73, 20–26] has been extended to (a) the quantitation of four other macromolecules: vz. streptococcal lipoteichoic acid, I blood group substance, desialylated bovine submaxillary mucin, and desialylated pig submaxillary mucin; and (b) the use of three other lectins: vz. wheat germ agglutinin, soybean agglutinin and peanut agglutinin. In all cases stable-affinity precipitin rockets were observed the heights of which bore an approximately linear relationships to the amount of sample analyzed. For all lectins, the detection limits were in the range of 15–25 ng. Furthermore, a new technique has been introduced called crossed affinoelectrophoresis which is basically a two-dimensional variant of rocket affinoelectrophoresis. This technique can be used with concanavalin A, wheat germ agglutinin, soybean agglutinin, or peanut agglutinin in the affinity gel and allows the examination of glycoprotein homogeneity. Modification of this technique, involving the use of other lectins or antiserum in intermediate gels, is also described and evaluated.     

Determination of neuraminidase-susceptible and total N-acetylneuraminic acid in cells by selected ion monitoring.

N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ($\text{m}\text{e}\text{= 814}$) and neuraminic acid β-methylglycoside (internal standard, $\text{m}\text{e}\text{= 714}$) in a gas chromatograph—mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of $\text{0.3μ}\text{mol}\text{10}{}^9$ cells. NANA was quantified using as few as 5 × 10⁴ cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 10⁷ cells.     

High-performance liquid chromatographic assay for the determination of the novel etoposide derivative dimethylaminoetoposide (NK611) and its metabolites in urine of cancer patients

A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C₁₈ column with a mobile phase of acetonitrile-0.05 M KH₂PO₄ (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1–3.4% and 1.9–2.4% for all analytes and the accuracy was 101–107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I–IV in clinical pharmacokinetic investigations.     

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

The quantitation of sialyl-Tn (STn) conjugated to keyhole limpethaemocyanin (KLH) can be determined by quantitating the amountof ^N-acetylneuraminic acid (NANA) released by acid or enzymaticdigestion. An optimal 0.1 N H₂SO₄ acid hydrolysis at 80°Cresults in quantitative release of NANA with minimal loss. Arapid isocratic method for the quantitation and separation ofNANA is described using high-pH anion-exchange chromatographyand pulsed amperometric detection (PAD). Multiple injectionof NANA standard and/or samples containing protein led to adecrease in the PAD response which was corrected by additionof internal standard, -2-keto-3-deoxyoctonate (KDO). The ratioof NANA/KDO peak area or peak height gives a linear responsewith increasing amount of NANA in the range 2.5–20 µg/ml(r² = 0.99). The limit of quantitation (LOQ) for NANA usingthis isocratic method is 1.9 µg/ml ({small tilde}160 pmol/25µl injection). Based on the multiple determination theglycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w).Acid hydrolysis and the sialidase treatment of STn-KLH bothyielded a similar NANA content. The carrier protein, KLH, showedthe absence of NANA. The stability of glycoconjugate STn-KLHwas monitored by a gradient method which separated possibledegradation products STn-crotyl, NANA and GalNAc. Subjectingthe glycoconjugate STn-KLH to various stress conditions of temperature,pH and oxidation does not result in any release of sialic acid,GalNac and STn-crotyl group. high-pH anion-exchange chromatography mono-saccharide analysis pulsed amperometric detection sialyl-Tn stability of glycoconjugate     

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

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

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Determination of 13-cis-retinoic acid and its major metabolite, 4-oxo-13-cis-retinoic acid, in human blood by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10–2000 and 50–2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of clindamycin in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin

A simple, sensitive and specific liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C₁₈ column with a mobile phase of 80% acetonitrile–0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425→126 and 455→165 was used to quantitate I. A limit of quantitation of 0.0500 μg/ml was found. The assay exhibited a linear dynamic range of 0.0500–20.0 μg/ml and gave a correlation coefficient (r²) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 μg/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of −4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of −10.8 to −4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.     

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

High-performance liquid chromatographic measurement of urocanic acid isomers and their ratios in naturally light-exposed skin and naturally shielded skin

We have developed methods for sampling and extraction of trans-urocanic acid and cis-urocanic acid from human skin, and subsequent high-performance liquid chromatographic measurement of these isomers. Sampling involves applying cellophane adhesive tape to the skin for 10 s. Urocanic acid isomers were completely extracted by immersing the tape in KOH solution. The HPLC column was a Tosoh ODS 80_TS (250×4.6 mm I.D., 7 μm average particle size) eluted with 20 mM potassium dihydrogenphosphate containing 1 g/l sodium heptanesulphonate (pH 3.7)–acetonitrile (93:7, v/v) at a flow-rate of 1.0 ml/min. The isomers were detected by UV absorbance at 264 nm. This technique was used to analyze the ratio of trans-urocanic acid/cis-urocanic acid on human skin at various sites on the body. It was found that the ratio was low in naturally light-exposed skin and high in naturally shielded skin.     

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system

Extraction in a polyethylene glycol (PEG)–phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)–phosphate (6%) and PEG-4000 (14%)–phosphate (5.5%)–KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.     

Liquid chromatographic–mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthiritis. A robust, highly reliable and reproducible liquid chromatographic–mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C₁₈ column and chromatographed with a mobile phase comprised of acetonitrile–1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r²>0.994) over the concentration range 50–1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58–4.0% relative standard deviation and the accuracy is 99.4–107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC–MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.