Determination of vitamin A in dried human blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection

We have developed a high-performance capillary electrophoresis (HPCE) method to analyze the retinol (vitamin A) concentration as retinol-retinol binding protein (holo-RBP) from microvolumes of serum (5–10 μl) or one to two drops (∼20 μl) of blood collected and air-dried on blood collection filter paper. A 0.64-cm diameter disk was cut from the dried whole blood specimens and the samples were dissolved in a pretreatment buffer and filtered. Filtrate was injected onto the HPCE column for analysis. The separation was carried out in a 60 cm × 50 μm I.D. fused-silica capillary and the running voltage was 20 kV. A HeCd laser with a wavelength of 325 nm was used for excitation, and the fluorescence of the holo-RBP complex was monitored at 465 nm by a photodiode. A virtual linear relationship was obtained for the retinol concentrations between HPCE and HPLC for 28 serum samples, 19 dried venous blood samples and 9 capillary dried blood spot samples, indicating that valid measures of serum retinol can be obtained from one to two drops of capillary blood collected on filter paper. The absolute detection limit for retinol by HPCE is below 3 μg/l. The method is very useful for vitamin A level screening, especially for children and premature new-born babies.     

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids,retinol, and tocopherols in human serum

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 μm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2–7.3% and 3.6–12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.     

A direct microassay for serum retinol (vitamin A alcohol) by using size-exclusion high-pressure liquid chromatography with fluorescence detection

Serum retinol (bound to plasma retinol-binding protein, RBP) can be determined by direct injection of as little as 20 microliter of serum or plasma by using size-exclusion high-pressure liquid chromatography (SE-HPLC) with fluorescence detection. Toyo Soda TSK G-3000SW columns (0.75 X 7.5-cm guard column plus 0.75 X 30-cm analytical column) were eluted with 0.2 M NaCl/0.01 M phosphate buffer (pH 6.8) at 1 ml/min, with detection at 280 nm for protein elution. Fluorescence of the retinol-RBP complex was monitored with excitation at 334 nm (interference filter) and emission at 425 nm (long-pass filter). The retinol-RBP complex eluted as two peaks, the holo-RBP-transthyretin complex (apparent molecular weight 70,000) and holo-RBP (apparent molecular weight 9000). Identities of these peaks were established by immunodiffusion assay of the proteins and by extraction and analysis of retinol. Nonideal interactions with the column packing seem to be responsible for the low apparent molecular weight of holo-RBP. The first peak predominated when large volumes of serum (100 to 250 microliters) were injected, and the second when small volumes (5 to 50 microliters) were analyzed. The integrated area of the two fluorescence peaks due to retinol bound to RBP was proportional to the volume of a serum sample injected over the range 5 to 250 microliters.(ABSTRACT TRUNCATED AT 250 WORDS)     

血清游离精氨酸的快速检测

建立快速、准确的精氨酸定量检测方法。采用 6 30 0黄金系统氨基酸分析仪 ,在锂柱生理体液分析方法基础上 ,建立血清游离精氨酸 (ARG)快速测定方法。血清样本经磺基水杨酸沉淀蛋白后取上清液进行色谱分析 ,色谱柱为Beckman公司阳离子交换柱 (12cm× 4 .0mm) ;流动相为 2 0mmol·L- 1 柠檬酸锂水溶液 ,流速为 2 0ml·h- 1 ;比色波长 5 70nm。该法检测精氨酸浓度的线性范围为 5mg·L- 1 ～ 5 0mg·L- 1 ,相关系数 0 .99834,最低检测限 1mg·L- 1 ,重复性 :日内RSD 0 .4 0 % ,日间RSD 0 .5 5 % ,回收率 97.6 7%～ 10 0 .6 7% (平均值 99.0 7% ) ;整个实验过程耗时 2 8min。该法简便、快速、准确、可靠 ,适用于临床和科研工作。     

Use of eluent containing surfactant for the liquid chromatographic analysis of porphyrins by direct serum injection

A gradient elution reversed-phase high-performance liquid chromatographic method was developed for the direct serum injection analysis of porphyrins based on the use of eluent containing an anionic surfactant (sodium dodecyl sulfate) at a concentration below the critical micelle concentration to elute the serum proteins at the column void volume. Separation and detection performances were tested with a mixture of porphyrin standards containing uro-, heptacarboxylic-, hexacarboxylic-, pentacarboxylic-, copro-, zinc proto- and mesoporphyrin in a model serum consisting of 50 mg/ml bovine serum albumin. Average limit of detection is 0.06 pmol with a 10-μl injection volume using fluorimetric excitation at the Soret band of porphyrins. The utility of this method for the direct serum injection analysis of porphyrins in human serum was evaluated by investigating serum samples from individuals suffering from iron-deficiency anemia and breast cancer.     

Chromatographic analysis of endogenous retinoids in tissues and serum

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 microl serum or 200mg tissue, the limits of detection for all-trans-retinoic acid were 0.15ng/ml or 0.3ng/g, respectively. The corresponding values for retinol were 1.2ng/ml or 2.4ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.     

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.     

Rapid quantitative determination of fat-soluble vitamins and coenzyme Q-10 in human serum by reversed phase ultra-high pressure liquid chromatography with UV detection

We are presenting the first ultra-high pressure LC (UHPLC) method for rapid quantitative measurement of vitamin A, E (α- and γ-tocopherol), β-carotene and CoQ₁₀ from human serum. The chromatography was performed on Shield RP₁₈ UHPLC column with UV detection. The method was validated based on linearity, accuracy, matrix effects study, precision and stability. The calibration was linear over the following range: 0.09–10.0 for retinol and γ-tocopherol, 0.05–5 for β-carotene, 0.9–100 for α-tocopherol and 0.14–15 mg/L for CoQ₁₀. The limit of detection and quantitation for retinol, γ-tocopherol, β-carotene, α-tocopherol and CoQ₁₀ were as follows 0.07/0.024, 0.018/0.06, 0.004/0.12, 0.078/0.261, 0.008/0.028 mg/L. The recoveries were above 85%. The inter- and intra-assay precision was below 10%. Reference intervals were established for children and adults. Because of its low cost, extremely short analysis time (2 min) and excellent chromatographic reproducibility this UHPLC method can easily be adopted for high-throughput clinical and pharmacokinetics studies.     

Simultaneous measurement of serum probucol and lipid-soluble antioxidants.

A method is described for the simultaneous measurement of probucol, retinol, tocopherols, lycopene, and carotenes by reverse phase high performance liquid chromatography. A high sensitivity was achieved by use of a microbore column and by monitoring the effluent at the optimum wavelengths of each substance with a diode array detector. The detection limits were lycopene 0.5 ng; alpha-carotene, beta-carotene, and retinol 1 ng; probucol 2 ng; alpha-tocopherol and gamma-tocopherol 15 ng. The eluent was acetonitrile-water-tetrahydrofuran 81.3:5.7:13 (v/v/v) and the flow rate was 0.4 ml/min. Quantitation was performed by use of the four internal standards retinol acetate, 2-pentanone bis(3,5-di-tert)mercaptole, alpha-tocopherol acetate, and retinol palmitate, which resemble the respective analytes in structure and/or polarity. In order to attain a reproducible recovery of particularly the carotenes, the total lipid content of the samples had to be controlled by dilution of the sample before extraction. The coefficients of variation for between-day determinations of a serum pool were 3.8% for retinol, 4.5% for probucol, 11.2% for gamma-tocopherol, 4.5% for alpha-tocopherol, 10.4% for lycopene, 8.0% for alpha-carotene, and 7.0% for beta-carotene.     

Gas chromatography of retinol and α-tocopherol without derivatization

An improved gas chromatographic method for the analysis of retinol and α-tocopherol in biological samples is described. The use of cold on-column injection in combination with wall coated open tubular column gas chromatography eliminates thermal decomposition of vitamin A and yields efficient separations of fat-soluble vitamins (A, D₂, D₃, and E) without derivatization. Peak tailing was judged to be minimal. Vitamins were quantified by flame ionization detection responses down to 3.5 ng injected, and their identifies were confirmed using gas chromatography-mass spectrometry. Extracts of biological samples were saponified, and sterols were removed using digitonin-impregnated celite chromatography before analysis by gas chromatography and gas chromatography-mass spectrometry. Recoveries of vitamins from a test diet ranged from 89 to 103%.     

A chemiluminescence automatic analyser for the measurement of biological compounds

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L -glutamate dehydrogenase and L -glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.     

Sensitive flow-injection method with peroxyoxalate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum

A sensitive and rapid flow-injection analysis (FIA) of total choline-containing phospholipids (PLs) and a selective FIA method for the class assay of choline-containing PLs combined with preparative HPLC were described. The FIA method is based on peroxyoxaxalate chemiluminescence (PO-CL) detection of hydrogen peroxide enzymatically formed from choline-containing PL. The linear standard curves were obtained up to 1 nmol/20-μl injection (r>0.999) with the detection limits of 1.3–1.6 pmol at a signal-to-noise ratio of 2. The total amounts of choline-containing PLs in human serum were ranged from 1.63 to 3.19 mg/ml. The HPLC separation of choline-containing PLs was achieved with an aminopropyl-modified silica gel column using a mixture of acetonitrile-methanol-10 mM ammonium phosphate buffer pH 5.8 as eluent. The eluate corresponding to each choline-containing PL was collected, evaporated, dissolved in 0.1% Triton X-100 aqueous solution, and then injected into FIA system. The FIA method combined with preparative HPLC was applied to the assay of human serum.     

Liquid chromatographic determination of manidipine in serum with electrochemical detection

A highly sensitive and selective method for determining a dihydropyridine calcium antagonist, manidipine, by liquid chromatography using column switching with electrochemical detection was developed. Manidipine in serum was extracted by a rapid and simple procedure based on C₈ bonded-phase extraction and then silica extraction. Manidipine and nilvadipine as an internal standard were separated on a C₈ bonded-phase HPLC column and detected by high conversion efficiency amperometric detection at +0.7 V. Manidipine and nilvadipine (I.S.) were separated from an endogenous interference peak in serum and concentrated on a pre-column (C₁₈) by column switching using an isocratic mobile phase, and then the corresponding fractions were introduced to an analytical column with a C₈ stationary phase. Determination of manidipine was possible over the concentration range 0.5–10 ng/ml: the limit of detection was 0.3 ng/ml. The recovery of manidipine added to serum was 93.1–98.4% with coefficients of variation of less than 7.1%. The method is applicable to drug level monitoring in the serum of healthy volunteers treated with manidipine and to the analysis of pharmacokinetics.     

Determination of amikacin in microlitre quantities of biological fluids by high-performance liquid chromatography using 1-fluoro-2,4-dinitrobenzene derivatization

Pre-column derivatization of amikacin with 1-fluoro-2,4-dinitrobenzene in 25 μl of guinea pig plasma or human serum produced a stable chromophore which was measured by UV detection after rapid separation on normal-phase or reversed-phase high-performance liquid chromatography systems. The reversed-phase system, selected for routine analysis due to instability of the normal-phase column, consisted of an Ultrasphere-ODS C18 column preceded by a guard column, and used acetonitrile—water (68:32) as the mobile phase. A high degree of linearity was found in the range of 2—64 μg/ml with a coefficient of variation averaging less than 5%.     

Detection of a mixed-backbone oligonucleotide (GEM 231) in liver and tumor tissues by capillary electrophoresis

A simple, rapid and sensitive method has been developed and validated for the analysis of a mixed-backbone oligonucleotide (GEM 231) in tumor tissues. The analysis was performed using a capillary electrophoresis (CE) system with UV detection. An extended light path (bubble cell) capillary column of 64.5 cm (effective length 56 cm)×50 μm I.D. is used as the separation column. The optimized chromatographic conditions were background electrolyte: sodium borate buffer (60 mM, pH 9.1), electrokinetic injection: 10 s, applied voltage: 30 kV, detection at λ=210 nm. A linear relationship was observed between the peak area and the amount of GEM 231 in the range of 1.0–1000 μg/ml. The lower detection limit of the drug was 100 pg with an average recovery of about 75±5%. The inter-day and intra-day relative standard deviations were <10%. Assay validation studies revealed that CE method is reproducible and specific for the determination of GEM 231 in tissue homogenates with a run time of less than 5 min.     

Quantitative determination of cyclic polylactic acid oligomers in serum by direct injection liquid chromatography tandem mass spectrometry

Polylactic acid (PLA) is a biodegradable polymer, currently used in pharmaceutical and surgical devices. There is a concern that cyclic polylactic acid (CPLA), which is a by-product of PLA synthesis, may be introduced into the human body as an undesirable contaminant. We carried out a quantitation investigation of the CPLA heptamer (CPLA-7) by liquid chromatography mass spectrometry (LC-MS). We found that CPLA-7 binds strongly with serum proteins and that only 62% of CPLA-7 was recovered after routine deproteination; therefore, we directly injected serum into the LC-MS/MS system after passage through a bovine serum albumin (BSA)-coated chromatographic column and found the recovery of CPLA-7 was improved to 84%, and that the detection (S/N=3) and quantitation limit (S/N=10 and below 15% relative standard deviation) were 1.5 and 2.5 ng/mL, respectively. We conclude that direct injection LC-MS/MS, using a BSA column, is a simple and effective quantitative analysis method for CPLA in serum.     

Rapid determination of ethylene glycol at toxic levels in serum and urine

The rapid gas chromatographic detection and determination of ethylene glycol in biological fluids is described. Phenylboronic acid in acetone was used for the esterification of glycol. The phenylboronates of ethylene glycol and 1,2-propylene glycol are not separated on a packed column of medium polarity (OV-17), but they can be separated on a non-polar column (OV-101). In both instances 1,3-propylene glycol can be used as an internal standard. The method requires only 100 μl of serum or urine and is suitable for trace analysis in an emergency toxicological laboratory. The utility of the method is demonstrated on two cases of human intoxication with ethylene glycol.     

Rapid separation of human globin chains in normal and thalassemia patients by RP-HPLC

The present study describes a rapid and sensitive high-performance liquid chromatography (HPLC) method for the detection of human globin chains in blood. The method involves direct injection of globin chains which prepared by a standard method onto a micro bondapack C18 reversed-phase column (7.8 mm I.D.) with UV detection at 280 nm. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 ml of fresh whole blood. We report here the rapid procedure for globin chain analysis. The present method will be useful for the determination of globin chain analysis in clinical laboratories, as well as in thalassemia and sickle cell disease patients.     

Analytical platform for verification and quantitation of target peptides in human serum: application to cathelicidin

A selective and sensitive, fully automated platform for verification and quantitative determination of target peptides in biofluids is proposed and then validated by development of a method for analysis of cathelicidin in human serum. The method is based on the on-line coupling of solid-phase extraction (SPE) and tandem mass spectrometry with direct infusion. Mass spectrometry analysis was carried out by multiple reaction monitoring using three transitions (one for quantitative analysis and two for qualitative analysis), all them confirmed by in silico fragmentation of the target peptide. Samples were prepared in the SPE workstation on a polymeric divinylbenzene resin by preconcentration, deproteinization, and cleanup, removing salts and interferences after direct injection of human serum. The analytical process required 12 min. The limits of detection and quantitation were 2.5 and 8.25 μg/L, respectively (0.20 and 0.66 pg on column). Repeatability and within-laboratory reproducibility were 2.4% and 2.7%, respectively. A dual-cartridge configuration was used to test recovery of cathelicidin in serum, resulting in 80%. Because quantitative retention in the cartridge was assessed, determination of cathelicidin was validated without using synthetic peptides labeled with stable isotopes. The hyphenated system allows full automation, thereby improving reproducibility and accuracy, as demanded by clinical analysis.     

An assay for phentolamine using high performance liquid chromatography with electrochemical detection

A new method is presented for the detection of phentolamine by high performance liquid chromatography with electrochemical detection. The electrochemical detector was used in the oxidative mode at +900 mV potential versus Ag/AgCl reference. The on-column detection limit for phentolamine using this method was 3 ng, and detector response was linear for 3-1000 ng injected on column. The coefficient of variation for replicate injections was 2.4%. The measurement of phentolamine in biological samples was accomplished using yohimbime as the internal standard; retention time for yohimbine was 3.0 min while phentolamine eluted at 4.75 min. Biological samples were buffered to pH 9.2 and extracted with diethyl ether, followed by back extraction into 0.1 N HCl. The extraction efficiency for this method was 99.4% for phentolamine in serum and 59.3% in liver tissue. The detection limit for phentolamine was 5 ng/ml for 1.0-ml serum samples, and was 10 ng/ml for 1.0-ml liver homogenate samples. The disappearance of phentolamine from serum and liver after administration of a single ip dose of phentolamine to mice was determined using this method. Absorption from the ip route was rapid, with peak phentolamine concentrations achieved in 15 min or less. The elimination half-life of phentolamine in serum was approximately 50 min and was paralleled by disappearance of phentolamine in the liver.