Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of paracetamol and guaifenesin in human plasma

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether-dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05-20.0 microg/ml for paracetamol and 5.0-2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within +/-2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.     

Fast concurrent determination of guaifenesin and pholcodine in formulations and spiked plasma using first derivative synchronous spectrofluorimetric approach

Guaifenesin and pholcodine are frequently co-formulated in certain dosage forms. A new fast first derivative synchronous spectrofluorometric method has been used for their simultaneous analysis in mixtures. Here, first derivative synchronous spectrofluorometry enabled the successful simultaneous estimation of guaifenesin at 283 nm and pholcodine at 275 nm using a wavelength difference (Δλ) of 40 nm. The method was fully validated following International Council of Harmonization guidelines. For guaifenesin and pholcodine, linearity was determined within the corresponding ranges of 0.05–0.30 and 0.10–6.0 μg/ml. The two drugs were effectively analyzed using the developed approach in their respective formulations, and the results showed good agreement with those attained using reference methods. The method demonstrated excellent sensitivity, with detection limits down to 0.007 and 0.030 μg/ml and quantitation limits of 0.020 and 0.010 μg/ml for guaifenesin and pholcodine, respectively. Therefore, the procedure was successful in determining these drugs simultaneously in vitro in spiked plasma samples and syrup dosage form. The developed methodology also offered an environmentally friendly advantage by utilizing water as the optimal diluting solvent throughout the whole work. Different greenness approaches were investigated to ensure the method’s ecofriendly properties.     

Validation of a high-performance liquid chromatographic method for the determination of doxycycline in turkey plasma

A high-performance liquid chromatographic method for the analysis of doxycycline in turkey plasma samples using demeclocycline hydrochloride as the internal standard was developed, optimized and validated, A one-step extraction procedure and an isocratic HPLC method with UV detection were used. No interferences with endogenous compounds or with the anticoagulant were observed, Linear calibration curves (r²>0.99) were obtained in water and plasma between 0 and 600 μg ml⁻¹. Good recoveries for doxycycline (>66%) and demeclocycline (>72%) were seen both in water and in plasma, The coefficient of variation was <9.86% for within-day reproducibility and <7.53% for the between-day reproducibility. The deviation between the mean value found and the true value was <14.5% (accuracy). The limit of detection was 0.1 μg ml⁻¹ in plasma samples. A good stability of doxycycline was observed in water and in plasma samples after storage for six months at −20°C (recovery >91%).     

Liquid chromatographic method for the determination of ticlopidine in human plasma

A simple high-performance liquid chromatographic method for determination of ticlopidine in human plasma using ultra violet detection was developed. The separation of the investigated compound and internal standard was achieved on a C₁₈ BD column with a 0.01 M potassium dihydrogen phosphate buffer (pH 4)–acetonitrile–methanol (20:40:40, v/v) mobile phase. The detection was performed at 215 nm. The compounds were isolated from plasma by Bond Elut C₁₈ solid-phase extraction, the mean absolute recovery was 84.9%. The limit of quantitation was 10 ng ml⁻¹, the limit of detection was 5 ng ml⁻¹. The bioanalytical method was validated with respect to linearity, within- and between-day accuracy and precision, system suitability and stability. All validated parameters were found to be within the internationally required limits. The developed analytical method for ticlopidine was found to be suitable for application in pharmacokinetic studies and human drug monitoring.     

Flow‐injection chemiluminescence determination of melamine in urine and plasma

A novel flow‐injection chemiluminescence method for the determination of melamine in urine and plasma was developed. It was found that melamine can remarkably enhance chemiluminescence emission from the luminol–K₃Fe(CN)₆ system in an alkaline medium. Under the optimum conditions, chemiluminescence intensity had a good linear relationship with the concentration of melamine in the range 9.0 × 10^–9–7.0 × 10^–6 g/mL, with a correlation coefficient of 0.9992. The detection limit (3σ) was 3.5 ng/mL. The method has been applied to determine the concentration of melamine in samples using liquid–liquid extraction. Average recoveries of melamine were 102.6% in urine samples and 95.1% in plasma samples. The method provided a reproducible and stable approach for the sensitive detection of melamine in urine and plasma samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous determination of guaifenesin enantiomers and ambroxol HCl using 50‐mm chiral column for a negligible environmental impact

Chiral stationary phases are conveniently used for enantiomeric separation of drugs by liquid chromatography. Consumption of large volumes of hazardous solvents is considered as a common challenge for the sustainability of this technique. To this end, a columnar chromatography has been adopted using 50‐mm‐length stationary phases. The study comprised five Phenomenex Lux cellulose‐ and amylose‐based columns for the separation of guaifenesin (GUA) enantiomers. In addition, an experimental design was used to optimize the gradient profile for the separation of racemic GUA and ambroxol HCl (AMB) binary mixture. The chromatographic method was achieved using Lux Cellulose‐1 (50 × 4.6 mm) as a chiral stationary phase and ethanol/water as a mobile phase with linear gradient elution of 20% to 70% ethanol in 6 minutes at a flow rate of 1.0 mL min^?1 and UV detection at 270 nm. Linearity ranges were found to be 50 to 1000 μg mL^?1 and 15 to 450 μg mL^?1 for each GUA enantiomer and AMB, respectively. Environmental, health and safety tool was used to assess and compare greenness of the proposed and reported methods. Short column indeed reduces the environmental impact by decreasing waste by about 60% and utilizing only 1‐mL ethanol in the mobile phase. The proposed method is a safer alternative for the simultaneous determination of drugs in their combined pharmaceutical formulation. The method has been validated and compared favorably with a reported one.     

High-performance liquid chromatographic determination of 4-methylpyrazole in plasma and in dialysate

A rapid method for the determination of 4-methylpyrazole (4-MP) levels in plasma and in dialysate by isocratic reversed-phase high-performance liquid chromatography with UV detection is described. The internal standard was the 3-methylpyrazole (3-MP). Plasma sample preparation consisted of a protein precipitation. Dialysate samples were injected without preparation. The method was linear up to 30 mg l⁻¹ in plasma and up to 5 mg l⁻¹ in dialysate. The within-day precisions (C.V.) were less than 4% in plasma and were less than 2% in dialysate. The day-to-day precisions (C.V.) were less than 7% in plasma and were less than 3% in dialysate. This method is easy to perform and has practical interest for clinicians who need to monitor in emergency 4-MP levels in ethylene glycol and methanol poisonings.     

Simultaneous high-performance liquid chromatographic determination of caffeine and theophylline for routine drug monitoring in human plasma

An HPLC method for the simultaneous determination of both caffeine and theophyllie in human plasma is described, using a reversed-phase chromatography column, heated by a thermostatic oven at 35°C, with UV detection and isocratic elution. The linearity and reproducibility of the method are verified. For the two drugs, the limit of detection is 0.1 μg ml⁻¹. This analytical method is rapid and reliable and allows routine controls of therapeutic levels of theophylline and caffeine, especially in premature infants where the volume of plasma samples is very small.     

Ultrafiltration-light absorption spectrophotometric determination of silicon in blood

An ultrafiltration-light absorption spectrometric method for soluble molybdate-reactive silicon was assessed and applied to bovine and ovine blood plasma and sera, giving precise analytical results. Interfering protein above molecular weight 10,000–25,000 was removed by ultrafiltration, and silicon in ultrafiltrates was quantitated by measuring light absorption at 810 nm of the 1,2,4-aminonaphthol sulfonic acid/ascorbic acid-reduced silicomolybdate. Chemical interferences on the color-forming reaction of remaining blood components were tested by measuring recoveries of silicon added to real blood plasma samples and to synthetic blood plasma solutions, the latter containing typical levels of the major ions Na⁺, K⁺, Ca²⁺, HCO₃^?, and Cl^?, together with varying quantities of the potential interferants (amount per analytical reaction): phosphate (0–0.5 mg P), ferric ion (0–3 mg), fluoride (0–1.25 mg), vanadate (0–0.5 mg V), arsenate (0–10 μg As), and germanate (0–0.5 μg Ge). The mean recovery of added 0.8–9 μg silicon/g of bovine and ovine plasma was 97.7% (SE = 1.0, n = 17); the mean recovery of 1 and 5 μg silicon from synthetic blood plasma solutions with interferant levels up to 50-fold that in normal plasma was 99.2% (SE = 0.3, n = 47). Silicon concentrations found in bovine and ovine blood plasma and sera were typically around 7 μg/ml with procedural reagent blanks consistently low at a mean of 0.12 μg/test (SD = 0.011, n = 20). The silicon level in Center for Disease Control bovine serum (reference specimen Lot R-2274) was found to be (mean ± SE, n = 10) 1.147 ± 0.013 μg/g or 1.172 ± 0.013 μg/ml (25°C). The method detectivity (detection limit) was estimated at 0.03 μg.     

High-resolution liquid chromatographic method using ultraviolet detection for determination of ondansetron in human plasma

This paper describes a simple technique for extraction and a sensitive high-performance liquid chromatographic method for separation and quantitation of ondansetron in human plasma. The procedure involved liquid–liquid extraction of ondansetron from plasma, reversed-phase HPLC separation and ultraviolet detection at 305 nm. The internal standard method was applied for quantitation. The recovery of ondansetron was >85%. Linearity was good throughout the concentration range anticipated in human plasma from investigations in panic disorder (0.5–15 ng/ml, r² ranging from 0.9953 to 0.9988). This method was applied to the determination of plasma concentrations of ondansetron in humans.     

Quantitative determination of cefepime in plasma and vitreous fluid by high-performance liquid chromatography

An isocratic reversed-phase HPLC method was developed to determine cefepime levels in plasma and vitreous fluid. Cefepime and the internal standard cefadroxil were separated on a Shandon Hypersil BDS C18 column by using a mobile phase of 25 mM sodium dihydrogen phosphate monohydrate (pH 3) and methanol (87:13, v/v). Ultraviolet detection was carried out at 270 nm. The retention times were 4.80 min for cefepime and 7.70 min for cefadroxil. This fast procedure which involves an efficient protein precipitation step (addition of HClO₄), allows a quantification limit of 2.52 μg ml⁻¹ and a detection limit of 0.83 μg ml⁻¹. Recoveries and absolute recoveries of cefepime from plasma were 96.13–99.44% and 94–102.5% respectively. The intra-day and inter-day reproducibilities were less than 2% for cefepime at 10, 30, 50 μg ml⁻¹ (n=10).The method was proved to be suitable for determining cefepime levels in human plasma and was modified to measure vitreous fluid samples.     

A novel spectrofluorimetric method for determination of imatinib in pure,pharmaceutical preparation,human plasma,and human urine

The following paper represents a simple, highly sensitive, responsive validated and developed spectrofluorimetric method for estimation of imatinib (IMB) in its pure, commercial preparation, human urine and human blood plasma. The calibration curve was in the range 4–900 ng ml^?1 for pure form and urine and 8–900 ng ml^?1 for plasma in a medium contains carboxymethyl cellulose (CMC) and acetate buffer (pH 5) with excitation wavelength (λ_ex) 230 nm and emission wavelength (λ_em) 307 nm. The limit of detection (LOD) was 0.37 ng ml^?1 for the pure form, 0.64 ng ml^?1 for human urine, and 0.70 ng ml^?1 for human plasma, while the limit of quantitation (LOQ) was 1.2 for pure form, 1.91 for urine and 2.1 for plasma. The suggested method was successfully applied for evaluation of IMB in tablets within 99% mean percentage recovery. The excipients that are usually used as additives in pharmaceutical dosage form did not interfere with the suggested method. The method was efficiently used for estimation of IMB in human urine and human plasma. The effect of some cations that might be present in urine and plasma was also studied. The method was also focused on human volunteers and in vitro drug release.     

High-performance liquid chromatographic determination of diazepam in plasma of children with severe malaria

A sensitive, selective and reproducible reversed-phase HPLC method with ultraviolet detection was developed for the quantification of diazepam in small plasma samples from children with severe malaria. The method involves plasma deproteinization with acetonitrile, followed by liquid–liquid extraction with ethyl acetate–n-hexane. Diazepam was eluted at ambient temperatures from a reversed-phase C₁₈ column with an acidic (pH 3.5) aqueous mobile phase (10 mM KH₂PO₄–acetonitrile, 69:31, v/v). Calibration curves in spiked plasma were linear from 10 to 200 ng (r²≥0.99). The limit of detection was 5.0 ng/ml, and relative recoveries at 25 and 180 ng were >87%. Intra- and inter-assay relative standard deviations were <15%. There was no interference from drugs commonly administered to children with severe malaria (phenobarbitone, phenytoin, chloroquine, quinine, sulfadoxine, pyrimethamine, halofantrine, cycloguanil, chlorcycloguanil, acetaminophen and salicylate). This method has been used for monitoring plasma diazepam concentrations in children with seizures associated with severe malaria.     

High-performance liquid chromatographic method for sensitive determination of the alkylating agent CB1954 in human plasma

A high-performance liquid chromatography (HPLC) method is described for the measurement of the weak alkylating agent CB1954 in human plasma. CB1954 can be used as an innocuous prodrug designed for activation by bacterial nitroreductases in strategies of gene-directed enzyme–prodrug therapy, and becomes activated to a potent bifunctional alkylating agent. The HPLC method involves precipitation and solvent extraction and uses Mitomycin C (MMC) as an internal standard, with a retention time for MMC of 5.85±0.015 min, and for CB1954 of 10.72±0.063 min. The limit of detection for CB1954 is 2.9 ng/ml, and this compares favourably with systems involving direct analysis of plasma (limit of detection 600 ng/ml, approximately). The method is now being used for pharmacokinetic measurements in plasma samples from cancer patients entering phase I clinical trials of CB1954. Results using serial plasma samples from one patient are presented. The patient was treated intravenously with CB1954 (6 mg/m²), and plasma clearance of the drug showed biphasic kinetics with α half-life 14.6 min, and β half-life 170.5 min.     

Enantioselective high-performance liquid chromatographic determination of nicardipine in human plasma

A sensitive method for the enantioselective high-performance liquid chromatography (HPLC) determination of nicardipine in human plasma is described. (+)-Nicardipine, (−)-nicardipine and (+)-barnidipine as an internal standard are detected by an ultraviolet detector at 254 nm. Racemic nicardipine in human plasma was extracted by a rapid and simple procedure based on C₁₈ bonded-phase extraction. The extraction samples were purified and concentrated on a pre-column using a C₁ stationary phase and the enantiomers of nicardipine are quantitatively separated by HPLC on a Sumichiral OA-4500 column, containing a chemically modified Pirkle-type stationary phase. Determination of (+)- and (−)-nicardipine was possible in a concentration range of 5–100 ng ml⁻¹ and the limit of detection in plasma was 2.5 ng ml⁻¹. The recoveries of (+)- and (−)-nicardipine added to plasma were 91.4–98.4% and 93.3–96.7%, respectively, with coefficients of variation of less than 9.0 and 9.4% respectively. The method was applied to low level monitoring of (+)- and (−)-nicardipine in plasma from healthy volunteers.     

Improved method for the determination of serotonin in plasma by high-performance liquid chromatography using on-line sample pre-treatment

An improved method for the determination of serotonin in platelet-rich plasma (PRP) and platelet-poor plasma (PPP), by reversed-phase high-performance liquid chromatograpy with electrochemical detection and direct plasma injection, is described. The chromatographic system comprises a strong cation-exchange pre-column and a C₁₈ analytical column. The method is selective, rapid, simple and sensitive, and offers good reproducibility and recovery. Reference values for serotonin concentrations in healthy adults (n = 10) are 31 nM for PPP and 6 nmol per 10⁹ platelets for PRP. The conditions used for the preparation of PRP and PPP may influence the serotonin concentration in PRP.     

High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine

A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10⁻⁷M.     

On-line chemiluminescence determination of mitoxantrone by capillary electrophoresis

A novel capillary electrophoresis (CE) with chemiluminescence (CL) detection method for the determination of mitoxantrone (MTX) has been developed, which based on the CL reaction of potassium ferricyanide with luminol in sodium hydroxide medium sensitized by MTX. Under optimum analytical conditions, MTX is determined over the range of 7.0 × 10⁻⁸–1.0 × 10⁻⁶ M with a detection limit of 1.0 × 10⁻⁸ M. The relative standard deviation (RSD) was 3.7%, 2.6% and 3.0% for 7.0 × 10⁻⁸, 5.0 × 10⁻⁷ and 1.0 × 10⁻⁶ M MTX (n = 11), respectively. In laboratory-built CE–CL apparatus, the proposed method has been applied to determination of MTX in commercial drug and spiked in human urine and plasma with satisfactory results.     

A rapid,sensitive method for the determination of 3-methylhistidine levels in urine and plasma using high-pressure liquid chromatography

A method is presented for the precolumn derivatization and subsequent high-pressure liquid chromatographic separation of 3-methylhistidine from urine and plasma. The solvent system is 10 mm sodium phosphate (pH 7.5) and acetonitrile. The elution can be performed isocratically and requires less than 10 min. Both fluorescent and ultraviolet detection may be utilized. This method is at least 10³ times more sensitive than conventional ion-exchange chromatography using ninhydrin. 3-Methylhistidine determinations performed on plasma and urine samples from normal volunteers correlated well with published literature values.     

Sensitive high-performance liquid chromatographic method for the determination of N-hexadecyl- and N-octadecyl-1-ß- -arabinofuranosylcytosine in plasma and erythrocytes

N⁴-Hexadecyl- and N⁴-octadecyl-1-ß- -arabinofuranosylcytosine (NHAC, NOAC) are two new cytostatic derivatives of cytosine arabinoside (ara-C) with improved cytostatic activity and stability against deamination. A high-performance liquid chromatography (HPLC) method was developed for the specific determination of NHAC and NOAC in plasma and erythrocytes, after solid-phase extraction using UV detection at 275 nm. Because of the strong binding of the drugs to proteins and membranes, the samples have to be pretreated with urea (plasma) or butanol and ultasonication (erythrocytes). The calibration curves are linear for both drugs (r> 0.999) in the concentration ranges 20–2100 μg/l for plasma and 40–4200 μg/l for erythrocytes, respectively. The within-day and between-day precision studies showed a good reproducibility, with coefficients of variation below 8.5%. The recoveries of the lipophilic ara-C derivatives are greater than 66%. The method described can be applied to pharmacokinetic studies with NHAC and NOAC.