Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

Determination of free urinary cortisol in cushing's syndrome using reversed-phase high performance liquid chromatography

Determination of free urinary cortisol is a test of choice in the diagnosis of Cushing's syndrome. In this study, cortisol was quantified using reversed-phase high-performance liquid chromatography (RP-HPLC) in urine samples previously extracted with ether and using triamcinolone acetonide as internal standard (IS). A BDS-Hypersil-C18® column, water-acetonitrile (72:28; v/v), with a flow rate of 1.0 mL/min and detection at 243 nm were used. This method showed to be both effective and efficient, with sensitivity and linearity ranging from 2.50 to 150 μg/L, and can be used in substitution to the radioimmunoassay technique within this concentration range.

Development and validation of a dissolution test with reversed-phase liquid chromatography analysis for rupatadine in tablet dosage forms

A dissolution test for in vitro evaluation of tablet dosage forms containing 10 mg of rupatadine was developed and validated by RP-LC. A discriminatory dissolution method was established using apparatus paddle at a stirring rate of 50 rpm with 900 mL of deaerated 0.01 M hydrochloric acid. The proposed method was validated yielding acceptable results for the parameters evaluated, and was applied for the quality control analysis of rupatadine tablets, and to evaluate the formulation during an accelerated stability study. Moreover, quantitative analyses were also performed, to compare the applicability of the RP-LC and the LC-MS/MS methods.

Simultaneous determination of non-steroidal anti-inflammatory drugs in pharmaceutical formulations and human serum by reversed phase high performance liquid chromatography

A rapid and sensitive method using high performance liquid chromatography has been developed and validated for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in pharmaceutical formulations and human serum. Six NSAIDs including: naproxen sodium, diclofenac sodium, meloxicam, flurbiprofen, tiaprofenic and mefenamic acid were analyzed simultaneously in presence of ibuprofen as internal standard on Mediterranea C18 (5 µm, 250 x 0.46 mm) column. Mobile phase comprised of methanol: acetonitrile: H2O (60:20:20, v/v; pH 3.35) and pumped at a flow rate of 1 mL min-1 using 265 nm UV detection. The method was linear over a concentration range of 0.25-50 µg mL-1 (r² = 0.9999).

RNA analysis by ion-pair reversed-phase high performance liquid chromatography

Ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC) is presented as a new, superior method for the analysis of RNA. IP RP HPLC provides a fast and reliable alternative to classical methods of RNA analysis, including separation of different RNA species, quantification and purification. RNA is stable under the analysis conditions used; degradation of RNA during the analyses was not observed. The versatility of IP RP HPLC for RNA analysis is demonstrated. Components of an RNA ladder, ranging in size from 155 to 1770 nt, were resolved. RNA transcripts of up to 5219 nt were analyzed, their integrity determined and they were quantified and purified. Purification of mRNA from total RNA is described, separating mouse rRNA from poly(A)+ mRNA. IP RP HPLC is also suitable for the separation and purification of DIG-labeled from unlabeled RNA. RNA purified by IP RP HPLC exhibits improved stability.

On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

Reversed phase HPLC determination of tamoxifen in dog plasma and its pharmaco-kinetics after a single oral dose administration

The analytical method developed to evaluate tamoxifen in dog plasma samples was precise, accurate, robust and linear in the range of 5-200 ng/mL. The limits of detection and quantification were 0.981 ng/mL and 2.97 ng/mL, respectively. Besides, the intra-day precision and accuracy variations were 8.78 and 10.16%, respectively. Tamoxifen concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (lambda=280 nm). The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses. This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies.

Fluorimetric determination of intra- and extracellular free amino acids in the microalgae Tetraselmis gracilis (Prasinophyceae) using monolithic column in reversed phase mode

This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 mu mol/L) to 12% for Ile (0.10 mu mol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).

Comparison of ion pair and buffered mobile phase chromatography of haloacetic acids using direct and indirect UV and fluorescence detection

Reversed-phase high performance liquid chromatographic methods for the analysis of Haloacetic acids have been developed and compared to conventional direct detection methods. Haloacetic acids commonly found in drinking water, including monochloro-, dichloro-, bromo-, iodo- and trichloroacetic acids- have been studied. The ion pairing agent benzyltributylammonium ion was studied in detail using indirect UV and indirect fluorescence detection. Five different competing ions were evaluated to decrease analysis times and lower the detection limit by this new method. The direct detection method utilized an ammonium sulfate buffer and UV detection yielding a detection limit of 100 ppb. The indirect method developed has the advantage of being able to simultaneously analyze UV and non-UV absorbing ions and molecules but requires long equilibration times and demonstrated lower sensitivity than the direct method. ^

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

Determination of gatifloxacin in bulk and tablet preparations by high-performance liquid chromatography

A sensitive, precise, and specific high-performance liquid chromatographic (HPLC) method was developed for the assay of gatifloxacin (GATX) in raw material and tablets. The method validation parameters yielded good results and included the range, linearity, precision, accuracy, specificity, and recovery. It was also found that the excipients in the commercial tablet preparation did not interfere with the assay. The HPLC separation was carried out by reversed-phase chromatography on a C18 absorbosphere column (250 x 4.6 mm id, 5 pm particle size) with a mobile phase composed of acetic acid 50/o--acetonitrile-methanol (70 + 15 + 15, v/v/v) pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 287 nm. The calibration graph for GATX was linear from 4.0 to 14.0 mu g/mL. The interday and intraday precisions (relative standard deviation) were less than 1.05%.

Reversed phase HPLC determination of zidovudine in rat plasma and its pharmacokinetics after a single intranasal dose administration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of furanic aldehydes in sugarcane bagasse by high-performance liquid chromatography with pulsed amperometric detection using a modified electrode with nickel nanoparticles

A glassy carbon electrode chemically modified with nickel nanoparticles coupled with reversed-phase chromatography with pulsed amperometric detection was used for the quantitative analysis of furanic aldehydes in a real sample of sugarcane bagasse hydrolysate. Chromatographic separation was carried out in isocratic conditions (acetonitrile/water, 1:9) with a flow rate of 1.0 mL/min, a detection potential of -50 mV vs. Pd, and the process was completed within 4 min. The analytical curves presented limits of detection of 4.0 × 10(-7) mol/L and 4.3 × 10(-7) mol/L, limits of quantification of 1.3 × 10(-6) and 1.4 × 10(-6) mol/L, amperometric sensitivities of 2.2 × 10(6) nA mol/L and 2.7 × 10(6) nA mol/L for furfural and 5-hydroxymethylfurfural, respectively. The values obtained in this sample by the standard addition method were 1.54 ± 0.02 g/kg for 5-hydroxymethylfurfural and 11.5 ± 0.2 g/kg for furfural. The results demonstrate that this new proposed method can be used for the quick detection of furanic aldehydes without the interference of other electroactive species, besides having other remarkable merits that include excellent peak resolution, analytical repeatability, sensitivity, and accuracy.

Development of a comprehensive on-line multidimensional high performance column liquid chromatography system for protein and peptide mapping with integrated size selective sample fractionation

Aufbau einer kontinuierlichen, mehrdimensionalen Hochleistungs-flüssigchromatographie-Anlage für die Trennung von Proteinen und Peptiden mit integrierter größenselektiver ProbenfraktionierungEs wurde eine mehrdimensionale HPLC-Trennmethode für Proteine und Peptide mit einem Molekulargewicht von <15 kDa entwickelt.Im ersten Schritt werden die Zielanalyte von höhermolekularen sowie nicht ionischen Bestandteilen mit Hilfe von 'Restricted Access Materialien' (RAM) mit Ionenaustauscher-Funktionalität getrennt. Anschließend werden die Proteine auf einer analytischen Ionenaustauscher-Säule sowie auf Reversed-Phase-Säulen getrennt. Zur Vermeidung von Probenverlusten wurde ein kontinuierlich arbeitendes, voll automatisiertes System auf Basis unterschiedlicher Trenngeschwindigkeiten und vier parallelen RP-Säulen aufgebaut.Es werden jeweils zwei RP-Säulen gleichzeitig, jedoch mit zeitlich versetztem Beginn eluiert, um durch flache Gradienten ausreichende Trennleistungen zu erhalten. Während die dritte Säule regeneriert wird, erfolgt das Beladen der vierte Säule durch Anreicherung der Proteine und Peptide am Säulenkopf. Während der Gesamtanalysenzeit von 96 Minuten werden in Intervallen von 4 Minuten Fraktionen aus der 1. Dimension auf die RP-Säulen überführt und innerhalb von 8 Minuten getrennt, wobei 24 RP-Chromatogramme resultieren.Als Testsubstanzen wurden u.a. Standardproteine, Proteine und Peptide aus humanem Hämofiltrat sowie aus Lungenfibroblast-Zellkulturüberständen eingesetzt. Weiterhin wurden Fraktionen gesammelt und mittels MALDI-TOF Massenspektrometrie untersucht. Bei einer Injektion wurden in den 24 RP-Chromatogrammen mehr als 1000 Peaks aufgelöst. Der theoretische Wert der Peakkapazität liegt bei ungefähr 3000.