Simplex optimization of headspace factors for headspace gas chromatography determination of residual solvents in pharmaceutical products

The purpose of the present work was to find optimum conditions of headspace gas chromatography (HS-GC) determination of residual solvents which usually appear in pharmaceutical products. Two groups of solvents were taken into account in the present examination. Group I consisted of isopropanol, n-propanol, isobutanol, n-butanol and 1,4-dioxane and group II included cyclohexane, n-hexane and n-heptane. The members of the groups were selected in previous investigations in which experimental design and chemometric methods were applied. Four factors were taken into consideration in optimization which describe HS conditions: sample volume, equilibration time, equilibrium temperature and NaCl concentration in a sample. The relative GC peak area served as an optimization criterion which was considered separately for each analyte. Sequential variable size simplex optimization strategy was used and the progress of optimization was traced and visualized in various ways simultaneously. The optimum HS conditions appeared different for the groups of solvents tested, which proves that influence of experimental conditions (factors) depends on analyte properties. The optimization resulted in significant signal increase (from seven to fifteen times)

Grouping of residual solvents present in pharmaceuticals using experimental planning and chemometric methods

The main effects of six experimental factors on the efficiency of HS (headspace) extraction in headspace gas chromatography – flame ionization detector (HS-GC-FID) determination of twenty organic solvents routinely used in production of pharmaceuticals were obtained on the basis of the results of experiments carried out according to the Plackett-Burman factorial design. The effects were used as a basis for grouping the solvents into five groups, the solvents belonging to a group responded similarly to changes of HS conditions. To this end, visualization approaches were used as well as chemometric methods: cluster analysis (CA) and principal component analysis (PCA). Moreover, the most important HS experimental factors were selected for further optimization of the HS-GC determination procedure

Visualization of amphetamine and its analogues in TLC

Derivatisation followed by iodine azide reaction was employed for detection of amphetamines and its analogues in TLC. The derivatisation reaction with phenyl isothiocyanate took place directly on the TLC plate before the developing step. Afterwards, the plate was sprayed with a mixture of sodium azide and starch solution and then exposed to iodine vapour. The obtained limits of detection were compared with other commonly visualization techniques: UV, iodine vapour, Marquis and Simon’s reagents, ninhydrin, Fast Black K

Examination of distribution of trace elements in hair, fingernails and toenails as alternative biological materials : application of chemometric methods

The aim of this study was to find correlations between several studied elements and analyzed materials as well as the application and validation of an analytical method to determine trace elements in hair, fingernails and toenails of healthy volunteers (normal concentration). The method developed covers washing, mineralization and ICP- MS determination of 10 elements (Ca, Cd, Co, Cr, Cu, Fe, Mg, Ni, Pb and Zn) in hair and nails. Concentrations of the selected elements in hair, fingernails and toenails were measured for 24 women and 18 men. Furthermore, a chemometric approach (Principal Component Analysis, PCA) was employed to evaluate the correlations between concentrations of the elements in hair and nails and between these materials. Until now PCA has not been frequently applied in handling and interpretation of the results of analysis of biological materials. However, the results of the present investigation show the high potential of PCA in extraction of valuable information from analytical measurements. Additionally, PCA has become a useful tool for visualization of the obtained results. Moreover, the cluster analysis (CA) was used to group the samples according to gender, taking into account two different groups of elements: essential and toxic

Risk of All-Cause Mortality in HIV Infected Patients Is Associated with Clinical, Immunologic Predictors and the CCR5 Δ32 Deletion

OBJECTIVE: Investigation of the interplay between the CCR5 Δ32/wt genotype and demographic, epidemiological, clinical and immunological factors associated with mortality in the cART era. DESIGN: Longitudinal data from 507 HIV-infected patients following the Δ32 allele detection were analyzed. METHODS: Cumulative 15 years mortality was calculated using Kaplan-Meyer methodology. Hazard ratios were estimated using univariate Cox models. Basing on Akakie information criteria and statistical significance multivariate Cox model was constructed and effect plots presenting adjusted hazard ratio time-dependency were drawn. Analysis of the association of all-cause mortality and CCR5 Δ32/wt genotype prior to the antiretroviral treatment (cART) initiation (n = 507) and on the therapy (n = 422) was also performed. RESULTS: A mortality rate of 2.66 (CI 2.57-3.19) per 100 person-years was observed. Univariate analysis factors modifying the risk of death included the CCR5 genotype, gender, history of cART, AIDS diagnosis and also CD4 lymphocyte nadir, zenith, the latest CD4 count and stable levels >500 cells/µl. For multivariate analysis the following predictors were selected: CCR5 genotype (HR for wt/wt 2.53, CI 1.16-5.53, p = 0.02), gender (HR for males 1.91, 95%CI 1.1-3.36, p = 0.023), introduction of combined antiretroviral treatment (HR 4.85, CI 3.0-7.89, if untreated or treated <1 month, p<0.0001) CD4 count of 500 cells/µl for six months or more (HR 4.16, CI 1.95-8.88 if not achieved, p = 0.028), the latest CD4 count (HR 5.44, CI 3.39-8.74 for <100 cells/µl, p<0.0001) and history of AIDS (HR 1.69, CI 1.03-2.79, p = 0.039). Among untreated individuals the Δ32/wt genotype was associated with notably better survival (p = 0.026), while among cART treated individuals the Δ32 mutation did not correlate significantly with higher survival rates (p = 0.23). CONCLUSIONS: The Δ32 CCR5 allele is associated with a reduction of the risk of all-cause mortality in HIV (+) patients alongside clinical and immunologic predictors such as AIDS, history of cART, lymphocyte CD4 cell count and gender

Ocena częstości występowania mutacji genów BRAF, KRas oraz metylacji genu RASSF1A w wolu guzkowym na podstawie badania materiału cytologicznego uzyskanego drogą biopsji aspiracyjnej cienkoigłowej

  Introduction: Standard pre-operative diagnosis of nodular goitre is not always conclusive. The decision about nodular goitre surgery is increasingly based on molecular methods. The aim of the study was to determine BRAF T1799A mutation and KRas proto-oncogene mutation, and the analysis of RASSF1A promoter methylation level in cytological material obtained from FNAB specimens of thyroid nodules. Material and methods: The study population consisted of 85 women and 12 men. The study material was genomic DNA isolated from peripheral blood and thyroid bioptates. Pyrosequencing was used for the evaluation of RASSF1 methylation level. KRas mutation was investigated with Sanger sequencing. BRAF mutation was analysed by standard methods of real-time amplification detection (real-time PCR) with the use of specific starters surrounding the mutated site. Results: A significant positive correlation was demonstrated between mean methylation of four CpG islands of RASSF1A gene and thyroid tumour volume and its largest diameter (p &lt; 0.05). KRas mutation was not detected in any of the 97 patients. In 7/85 subjects (8.2%) BRAF mutation was observed. In 6/7 patients with BRAF mutation, FNAB of thyroid nodules confirmed a benign nature of the lesions; the material was non-diagnostic in one patient, and papillary thyroid cancer was diagnosed on the basis of postoperative histopathology assessment. Conclusions: The results of genetic tests reported in our study indicate that the presence of BRAF mutation or higher RASSF1A methylation levels in FNAB cytology specimens of benign lesions may be useful in the assessment of oncological risk, while the evaluation of KRas proto-oncogene mutation is not a valuable test in pre-operative diagnosis of nodular goitre. (Endokrynol Pol 2015; 66 (5): 384–393)    Wstęp: Na podstawie standardowej przedoperacyjnej diagnostyki wola guzkowego nie zawsze uzyskuje się jednoznaczne rozpoznanie. Coraz częściej w kwalifikacji wola guzkowego do zabiegu operacyjnego wykorzystywane są metody badań molekularnych. Celem pracy było oznaczenie mutacji T1799A genu BRAF i mutacji protoonkogenu KRas oraz analiza stopnia metylacji promotora genu RASSF1A w materiale komórkowym uzyskanym z guzków tarczycy na drodze biopsji aspiracyjnej cienkoigłowej. Materiały i metody: Badaniami objęto 85 kobiet i 12 mężczyzn. Materiał do badań stanowił genomowy DNA wyizolowany z krwi obwodowej pacjentów oraz z bioptatów tarczycy. Do oceny stopnia metylacji genu RASSF1 wykorzystano metodę pirosekwencjonowania. Mutacje genu KRas badano metodą sekwencjonowania Sangera. Do oznaczania mutacji BRAF użyto standardowej metodologii detekcji amplifikacji w czasie rzeczywistym (real-time PCR) z zastosowaniem specyficznych starterów otaczających miejsce zmutowane. Wyniki: Wykazano, że średnia metylacji czterech wysp CpG w genie RASSF1A znamiennie, dodatnio koreluje z objętością guza tarczycy i największym wymiarem guza (p &lt; 0,05). U żadnej z 97 osób nie stwierdzono mutacji Kras. U 7/85 badanych (8,2%) stwierdzono obecność mutacji genu BRAF. U 6/7 osób z obecnością mutacji BRAF, BAC guzków tarczycy wykazała łagodny charakter tych zmian, u jednej osoby otrzymano materiał niediagnostyczny, a na podstawie pooperacyjnego badania histopatologicznego rozpoznano raka brodawkowatego tarczycy. Wnioski: Otrzymane wyniki badań genetycznych wskazują, że obecność mutacji genu BRAF lub wyższego odsetka metylacji genu RASSF1A w materiale cytologicznym z biopsji aspiracyjnej cienkoigłowej zmiany cytologicznie łagodnej może mieć znaczenie w ocenie zagrożenia onkologicznego, podczas gdy ocena mutacji protonkogenu KRas nie jest przydatna w diagnostyce przedoperacyjnej wola guzkowego. (Endokrynol Pol 2015; 66 (5): 384–393)

Meeting the WHO 90% target : antiretroviral treatment efficacy in Poland is associated with baseline clinical patient characteristics

Introduction: Modern combined antiretroviral therapies (cART) allow to effectively suppress HIV-1 viral load, with the 90% virologic success rate, meeting the WHO target in most clinical settings. The aim of this study was to analyse antiretroviral treatment efficacy in Poland and to identify variables associated with virologic suppression. Methods: Cross-sectional data on 5152 (56.92% of the countrywide treated at the time-point of analysis) patients on cART for more than six months with at least one HIV-RNA measurement in 2016 were collected from 14 Polish centres. Patients’ characteristics and treatment type-based outcomes were analysed for the virologic suppression thresholds of <50 and <200 HIV-RNA copies/ml. CART was categorized into two nucleos(t)ide (2NRTI) plus non-nucleoside reverse transcriptase (NNRTI) inhibitors, 2NRTI plus protease (PI) inhibitor, 2NRTI plus integrase (InI) inhibitor, nucleos(t)ide sparing PI/r+InI and three drug class regimens. For statistics Chi-square and U-Mann Whitney tests and adjusted multivariate logistic regression models were used. Results: Virologic suppression rates of <50 copies/mL were observed in 4672 (90.68%) and <200 copies/mL in 4934 (95.77%) individuals. In univariate analyses, for the suppression threshold <50 copies/mL higher efficacy was noted for 2NRTI+NNRTI-based combinations (94.73%) compared to 2NRTI+PI (89.93%), 2NRTI+InI (90.61%), nucleos(t)ide sparing PI/r+InI (82.02%) and three drug class regimens (74.49%) (p < 0.0001), with less pronounced but significant differences for the threshold of 200 copies/mL [2NRTI+NNRTI-97.61%, 2NRTI+PI-95.27%, 2NRTI+InI-96.61%, PI/r+InI- 95.51% and 86.22% for three drug class cART) (p < 0.0001). However, in multivariate model, virologic efficacy for viral load <50 copies/mL was similar across treatment groups with significant influence by history of AIDS [OR:1.48 (95%CI:1.01–2.17) if AIDS diagnosed, p = 0.046], viral load < 5 log copies/mL at care entry [OR:1.47 (95%CI:1.08–2.01), p = 0.016], baseline lymphocyte CD4 count ≥200 cells/µL [OR:1.72 (95%CI:1.04–2.78), p = 0.034] and negative HCV serology [OR:1.97 (95%CI:1.29–2.94), p = 0.002]. For viral load threshold <200 copies/mL higher likelihood of virologic success was only associated with baseline lymphocyte CD4 count ≥200 cells/µL [OR:2.08 (95%CI:1.01–4.35), p = 0.049] and negative HCV status [OR:2.84 (95%CI:1.52–5.26), p = 0.001]. Conclusions: Proportion of virologically suppressed patients is in line with WHO treatment target confirming successful application of antiretroviral treatment strategy in Poland. Virological suppression rates depend on baseline patient characteristics, which should guide individualized antiretroviral tre0atment decisions