Simultaneous determination of antidementia drugs by HPLC-MS: validation data of the method and plasma level determinations in 300 patients

Aims: A rapid and simple HPLC-MS method was developed for the simultaneousdetermination of antidementia drugs, including donepezil, galantamine, rivastigmineand its major metabolite NAP 226 - 90, and memantine, for TherapeuticDrug Monitoring (TDM). In the elderly population treated with antidementiadrugs, the presence of several comorbidities, drug interactions resulting frompolypharmacy, and variations in drug metabolism and elimination, are possiblefactors leading to the observed high interindividual variability in plasma levels.Although evidence for the benefit of TDM for antidementia drugs still remains tobe demonstrated, an individually adapted dosage through TDM might contributeto minimize the risk of adverse reactions and to increase the probability of efficienttherapeutic response. Methods: A solid-phase extraction procedure with amixed-mode cation exchange sorbent was used to isolate the drugs from 0.5 mL ofplasma. The compounds were analyzed on a reverse-phase column with a gradientelution consisting of an ammonium acetate buffer at pH 9.3 and acetonitrile anddetected by mass spectrometry in the single ion monitoring mode. Isotope-labeledinternal standards were used for quantification where possible. The validatedmethod was used to measure the plasma levels of antidementia drugs in 300patients treated with these drugs. Results: The method was validated accordingto international standards of validation, including the assessment of the trueness(-8 - 11 %), the imprecision (repeatability: 1-5%, intermediate imprecision:2 - 9 %), selectivity and matrix effects variability (less than 6 %). Furthermore,short and long-term stability of the analytes in plasma was ascertained. Themethod proved to be robust in the calibrated ranges of 1 - 300 ng/mL for rivastigmineand memantine and 2 - 300 mg/mL for donepezil, galantamine and NAP226 - 90. We recently published a full description of the method (1). We found ahigh interindividual variability in plasma levels of these drugs in a study populationof 300 patients. The plasma level measurements, with some preliminaryclinical and pharmacogenetic results, will be presented. Conclusion: A simpleLC-MS method was developed for plasma level determination of antidementiadrugs which was successfully used in a clinical study with 300 patients.

A specific HPLC method for determination of beta-blockers topically used in ophthalmological diseases.

A HPLC method is presented for the identification and quantification in plasma and urine of beta-adrenergic receptor antagonists (betaxolol, carteolol, metipranolol, and timolol) commonly prescribed in ophthalmology. An extraction method is described using pindolol as an internal standard. An RSIL 10 micron column was used. The lower detection limits of the beta-blockers were found to be 4-27 ng/ml. This method is simple, rapid and sensitive; moreover, it allows the determination of 8 other beta-blockers.

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS.

A simple and sensitive LC-MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2-200 ng/mL for DUL, OLA, and STN, 4-200 ng/mL for DSTN, 5-1000 ng/mL for ARI, DARI and finally 2-1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

Screening for wound-induced oxylipins in Arabidopsis thaliana by differential HPLC-APCI/MS profiling of crude leaf extracts and subsequent characterisation by capillary-scale NMR

A simple non-targeted differential HPLC-APCI/MS approach has been developed in order to survey metabolome modifications that occur in the leaves of Arabidopsis thaliana following wound-induced stress. The wound-induced accumulation of metabolites, particularly oxylipins, was evaluated by HPLC-MS analysis of crude leaf extracts. A generic, rapid and reproducible pressure liquid extraction procedure was developed for the analysis of restricted leaf samples without the need for specific sample preparation. The presence of various oxylipins was determined by head-to-head comparison of the HPLC-MS data, filtered with a component detection algorithm, and automatically compared with the aid of software searching for small differences in similar HPLC-MS profiles. Repeatability was verified in several specimens belonging to different series. Wound-inducible jasmonates were efficiently highlighted by this non-targeted approach without the need for complex sample preparation as is the case for the 'oxylipin signature' procedure based on GC-MS. Furthermore this HPLC-MS screening technique allowed the isolation of induced compounds for further characterisation by capillary-scale NMR (CapNMR) after HPLC scale-up. In this paper, the screening method is described and applied to illustrate its potential for monitoring polar and non-polar stress-induced constituents as well as its use in combination with CapNMR for the structural assignment of wound-induced compounds of interest

Klooriheksidiinipitoisuus vesirokkovoiteessa - HPLC-menetelmän kehitys ja validointi

Tässä opinnäytetyössä kehitettiin HPLC-menetelmä klooriheksidiinipitoisuuden määrittämiseksi Professorin vesirokkovoiteesta. Menetelmä kehitettiin Yliopiston Apteekin analyyt-tiselle laboratoriolle. Klooriheksidiini on vesirokkovoiteessa käytetty vaikuttava aine. Se ehkäisee ja hoitaa ihon punoitusta ja turvotusta ja on nykyään tunnetuin antiseptinen aine. Klooriheksidiinipitoisuutta ei ole vesirokkovoiteesta tutkittu, eikä sen säilyvyys voiteessa ole tämän vuoksi varmaa. Yliopiston Apteekki määrittää vesirokkovoiteen käyttöiäksi kaksi vuotta. Opinnäytetyön tarkoituksena on selvittää, onko Yliopiston Apteekin määrittelemä kahden vuoden kestoaika vesirokkovoiteelle liian pitkä. Epäiltiin, että klooriheksidiini hajoaa vesirokkovoi-teessa parakloorianiliiniksi kahden vuoden aikana. Työn tarkoituksena oli kehittää HPLC-menetelmä klooriheksidiinipitoisuuden analysoimiseksi vesirokkovoiteesta. Työhön liittyi näytteenkäsittelyn ja uuttoliuottimen määritys vesirokkovoiteelle. Kirjallisuudessa esiintyviä menetelmiä testattiin ja muokattiin tähän työhön sopiviksi. Kun menetelmän kehitys oli saatu valmiiksi, uusi menetelmä validoitiin. Validoidun menetelmän avulla analysoitiin vanhoja erävarastonäytteitä, joiden avulla selvitettiin klooriheksidiinin säilyvyys voiteessa. Lopputulos oli, että klooriheksidiini säilyy voiteessa ainakin kolme vuotta hajoamattomassa muodossa. Voiteen valmistusprosessissa oli kuitenkin parannettavaa, koska klooriheksidiinipitoisuus vaihteli huomattavasti valmistuserien välillä. Klooriheksidiinipitoisuuden tutkimisen aloittaminen valmistettavista eristä oli tutkimustulosten mukaan aiheellista. Vesirokkovoiteen pitoisuuden tutkimisen seurauksena on suositeltavaa aloittaa lisäksi uusi säilyvyysseurantatutkimus, joka sisältää myös pitoisuustutkimukset.

Validació del mètode analític per la determinació d'aminoàcids i carbohidrats d'un producte farmacèutic per HPLC

Per poder desenvolupar un producte farmacèutic és necessari establir un mètode d’anàlisis que permeti determinar i quantificar totes aquelles substàncies que conté, ja sigui referent als principis actius; a les impureses i productes de degradació, conservants, antioxidants,... Grans entitats com la ICH remarquen la importància de validar els mètodes analítics ja que és la via per demostrar que aquell producte compleix les garanties de qualitat prèviament establertes. Així doncs, l’objectiu d’aquest Treball Final de Grau és poder desenvolupar i validar dos mètodes analítics per a la determinació d’aminoàcids i carbohidrats respectivament, d’un producte farmacèutic per cromatografia líquida (HPLC). Per tal de poder concloure que aquell mètode és adequat per la determinació per la qual ha estat desenvolupat, és necessari obtenir resultats que compleixin els criteris d’acceptació corresponents als paràmetres que han de ser avaluats en una validació analítica. Aquests paràmetres són: la precisió, la selectivitat, l’exactitud i la linealitat i el rang. Els resultats d’aquest projecte han demostrat que els dos mètodes desenvolupats són adequats per a la determinació de tres dels principis actius (aminoàcid 1, aminoàcid 2 i carbohidrat 1) que conté el producte farmacèutic d’ús veterinari analitzat; i poden ser validats ja que compleixen els criteris d’acceptació dels paràmetres avaluats que proposa la ICH. El mètode per la determinació de carbohidrats no és vàlid per el carbohidrat 2, ja que durant el desenvolupament es va detectar que una bona part d’aquest passava a carbohidrat 1 (desplaçament de l’equilibri ceto-enòlic que hi ha entre el carbohidrat 1 i el carbohidrat 2 a pHs alts). És per aquest motiu, que es pot concloure que aquest mètode no és vàlid i es recomana seguir investigant per a poder desenvolupar un mètode analític adient.

Extração por fluido supercrítico de alguns inseticidas carbamatos em amostras de batata, com determinação por HPLC/fluorescência e confirmação por HPLC/espectrometria de massas

Six supercritical fluid extraction (SFE) methods were tested, by varying the following operational parameters: CO2 pressure, time and temperature of extraction, type and proportion of static modifier, and Hydromatrix®/sample rate into cell. Firstly, insecticide carbamates were extracted from spiked potatoes samples (fortification level of 0,5 mg.Kg-1) by using SPE procedures, and then final extracts were analyzed HPLC/fluorescence. Good performance was observed with SFE methods that operated with values of temperature and CO2 pressure of 50 ºC and 350 bar, respectively. Best efficiency was obtained when it was used acetonitrile as a modifier (3% on the cell volume), and Hydromatrix®/sample rate of 2:1. Static time was of 1 min; total extraction time was of 35 min; dynamic extraction was performed with 15 mL of CO2, and it was used methanol (2 mL) for the dissolution of the final residue. In such conditions, pesticide recoveries varied from 72 to 94%, depending on the analyzed compound. In higher extraction temperatures, a rapid degradation was observed for some compounds, such as aldicarb and carbaryl; presence of their metabolites was further confirmed by HPLC-APCI/MS in positive mode. Detection limits for chromatographic analysis varied from 0,2 to 1,3 ng.

Determination of the HMX and RDX content in synthesized energetic material by HPLC, FT-MIR, and FT-NIR spectroscopies

A new method has been developed for determining the content of mixtures of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), the HMX/RDX ratio, in explosive compositions by Fourier transform infrared spectroscopy (FT-IR), in the regions MIR (mid infrared) and NIR (near infrared) with reference values obtained by chromatographic analysis (HPLC). Plots of relative MIR (A917 / A783) or NIR absorbance values (A4412 / A4317) versus HMX/RDX ratio determined by HPLC analysis revealed good linear relationships.

Comparison of GC and HPLC for quantification of organic acids in two jaboticaba (Myrciaria) fruit varieties

Gas chromatography (GC) with trimethylsilyl derivative formation was compared to high-performance liquid chromatography (HPLC) for quantification of organic acids (OAs) in two jaboticaba (Myrciaria) fruit (pulp and pericarp) varieties (Sabará and Açu Paulista). Succinic and citric acids were the major OAs found in all the samples analyzed. Besides being much more tedious, the results obtained with GC were significantly lower than HPLC (p<0.05) when the data (acids, variety, two parts and flowering days) were considered together. The presence of both acids was confirmed by gas chromatography-mass spectrometry (GC-MS).

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.

HPLC-ESI-MS/MS analysis of oxidized di-caffeoylquinic acids generated by metalloporphyrin-catalyzed reactions

This paper reports an HPLC-ESI-MS/MS investigation on the oxidation of 3,5- and 4,5- dicaffeoylquinic acid using iron(III) tetraphenylporphyrin chloride as catalyst. Two major mono-oxidised products of the quinic acid moiety have been identified for both compounds. However, only the 4,5-derivative afforded two different tri-oxo products. Thus, it seems that the oxidation pattern depends on the number and positions of the caffeic acid moieties present in caffeoylquinic acid molecules.

Rapid and accurate hplc-dad method for the determination of the herbicide bispyribac-sodium in surface water, and its validation

A new method is described for the determination of the herbicide bispyribac-sodium in surface water, especially from river and irrigated rice water samples. The method involves extraction in solid phase and quantification by high performance liquid chromatography with diode array detection (HPLC-DAD). After optimization of the extraction and separation parameters, the method was validated. The method presented average recoveries of 101.3 and 97.7%, under repeatability and intermediate precision conditions, respectively, with adequate precision (RSD from 0.9 to 7.5%). The method was applied for the determination of bispyribac-sodium in surface water samples with a limit of detection of 0.1 μg L-1.

Determination of cetirizine in tablets and compounded capsules: comparative study between CE and HPLC

A capillary electrophoresis (CE) method was developed and validated for determination of cetirizine dihydrochloride in tablets and compounded capsules. The electrophoretic separation was performed in an uncoated fused-silica capillary (40 cm x 50 μm i.d.) using 20 mmol L-1 sodium tetraborate buffer (pH 9.3) as background electrolyte, a hydrodinamic sample injection at 50 mBar for 5 s, 20 KV applied voltage at 25 °C, and detection at 232 nm. The proposed method was compared with the high performance liquid chromatographic (HPLC) method previously validated for this drug, and statistical analysis showed no significant difference between the techniques.

Losartan potassium dissolution test for drug release evaluation in pharmaceutical capsules using HPLC and UV spectrophotometry

This work describes the development and validation of a dissolution test for 50 mg losartan potassium capsules using HPLC and UV spectrophotometry. A 2(4) full factorial design was carried out to optimize dissolution conditions and potassium phosphate buffer, pH 6.8 as dissolution medium, basket as apparatus at the stirring speed of 50 rpm and time of 30 min were considered adequate. Both dissolution procedure and analytical methods were validated and a statistical analysis showed that there are no significant differences between HPLC and spectrophotometry. Since there is no official monograph, this dissolution test could be applied for quality control routine.

Development of new dissolution test and HPLC-RP method for anti-parasitic ornidazole coated tablets

This work aimed the development and validation of a new dissolution test for ornidazole coated tablets. The dissolution conditions were determined after testing Sink conditions, dissolution medium, apparatus, stirring speed, 24 h stability and medium filtration influence. The best conditions were paddle at a stirring speed of 75 rpm and 900 mL of 0.1 M HCl. A new HPLC quantification method was developed and validated. The dissolution test and quantification method showed to be adequate for their purposes and could be applied for quality control of ornidazole coated tablets, since there is no official monograph.