Detection of 14 sulfonate esters impurities of active pharmaceutical ingredients based on GC-MS/MS and LC-MS/MS / 药学学报

Two methods including gas chromatography tandem mass spectrometry (GC-MS/MS) and high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) were established to detect common alkyl sulfonates and aryl sulfonates genotoxic impurities. Four alkyl sulfonates and methyl benzenesulfonate were determined by GC-MS/MS using butyl methanesulfonate as the internal standard, the chromatographic column was HP-5MS UI (30 mm × 0.25 mm, 0.25 µm), the carrier gas was helium, the flow rate was 1.0 mL·min-1 in a constant flow mode, the sample inlet temperature was set to 250 ℃, the split ratio was 10∶1, and the initial temperature of the heating program was 80 ℃, maintained for 1 minute, and then increased to 240 ℃ at a heating rate of 30 ℃·min-1 for 2 minutes. The mass spectrometry detector was an electron bombardment ion source (EI source), the data collection condition was multi reaction monitoring mode (MRM), and method validation using the raw material of clinical drug citalopram hydrobromide as a sample. The results showed that the linear range of four alkyl sulfonates and methyl benzenesulfonate were good at 3-50 ng·mL-1 and 9-150 ng·mL-1, with a correlation coefficient of r > 0.999, The spiked recovery was 80%-120%. The detection limits were 1 and 3 ng·mL-1; Ten aryl sulfonates determined by LC-MS/MS, the chromatographic column was CSH Fluoro phenyl (100 mm × 2.1 mm, 1.7 µm), the mobile phase was methanol (B)-5 mmol·L-1 ammonium formate (D), with a flow rate of 0.2 mL·min-1, and gradient elution was performed. The gradient program (T/% B) was set as 0/20, 25/90, 35/90, 42/20. The mass spectrometer detector was electro spray ionization with positive ionization mode (ESI+), the data collection was in dynamic multi reaction monitoring mode (dMRM), and the method was validated using the raw material of the clinical drug citalopram hydrobromide as a sample. The results showed that the linear range of aryl sulfonates were good at 9-2 000 ng·mL-1, 3-100 ng·mL-1 and 0.9-30 ng·mL-1, respectively. The correlation coefficient r > 0.999, the spiked recovery was 80%-120%. The detection limits were 30, 1 and 0.3 ng·mL-1. Two detection methods did not detect potential sulfonate genotoxicity impurities in the above APIs. The established analytical methods are reliable and effective, which can provide reference for drug quality control and detection.

Simultaneous determination of 6 kinds of N-nitrosamines genotoxic impurities in losartan potassium raw material and its formulations by GC-MS/MS / 中国药房

OBJECTIVE To establish a method for simultaneous determination of the contents of 6 kinds of N-nitrosamines genotoxic impurities in losartan potassium raw material and its formulations. METHODS GC-MS/MS was adopted to determine 6 kinds of N-nitrosamines genotoxic impurities in losartan potassium raw material， Losartan potassium tablet， Losartan potassium capsule and Losartan potassium hydrochlorothiazide tablets， such as N-nitrosodimethylamine （NDMA）， N-nitrosodiethylamine （NDEA）， N-ethyl-N-nitroso-2-propanamine （NEiPA）， N-nitrosodiisopropylamine （NDiPA）， N-nitrosodipropylamine （NDPA） and N-nitrosodibutylamine （NDBA）. The separation was performed on SHIMADZU SH-L-17Sil MS capillary column by temperature- programmed GC， with injector temperature of 250 ℃ ， sample size of 1 μL， carrier gas of helium， and carrier flow rate of 1 mL/min. Electron ionization and multiple reaction monitoring （MRM） data acquisition mode were used， with an ion source temperature of 250 ℃ and solvent delay time of 3.1 min. RESULTS The separation among NDMA， NDEA， NEiPA， NDiPA， NDPA， NDBA and adjacent chromatographic peaks was good， and the separation rate was higher than 3.8； the linear ranges of them were 4.9-486.0， 4.9-488.5， 4.5-451.5， 6.8-683.5， 5.2-525.0 and 5.2-520.0 ng/mL（all r≥0.999 8）. The limits of quantitation were 4.86， 4.88， 4.52， 6.84， 5.25 and 5.20 ng/mL； the limits of detection were 0.97， 0.98， 0.90， 1.37， 1.05 and 1.04 ng/mL. RSDs of repeatability tests were 2.2%-5.6%（n＝6）， those of precision tests were 0.5%-1.4%（n＝6）， and those of stability tests were 1.5%-3.4%（n＝5）， respectively. Average recoveries of low-， medium- and high-concentration solution were 83.4%-103.0% （RSDs were 1.2%-6.3%， n＝3）， respectively. No one among the 6 kinds of N-nitrosamines genotoxic impurities was detected in both losartan potassium raw material and formulations. CONCLUSIONS The method is good in separation effect， highly accurate， sensitive and simple. It can be used in the determination of the 6 kinds of N-nitrosamines genotoxic impurities.

Catequinas del té verde: efectos antigenotóxicos y genotóxicos. Revisión sistemática / Green tea catechins: antigenotoxic and genotoxic effects. Systematic review

Las catequinas del té verde (Camellia sinensis) (CTV) presentan efectos benéficos para la salud asociados a su potencial antioxidante. Por otra parte, el estrés oxidante es una de las vías de inducción de daño genotóxico. De ahí que, en la presente revisión se realizó un análisis de los efectos antigenotóxicos y genotóxicos de las CTV, haciendo énfasis en las vías implicadas en estos procesos y sus efectos en la salud. Se realizó una revisión de artículos indexados en las bases de datos de PubMed® y Science Direct® (2021) con las palabras clave "green tea" y "green tea catechins". Se delimitaron los estudios utilizando los operadores booleanos "AND", "OR" y "NOT" ("antigenotoxic", "genotoxic", "antioxidant" y "prooxidant"). En su mayoría se consideraron las publicaciones del 2016 al 2021. Se observó que los efectos benéficos en la salud de las CTV están relacionados con: a) su actividad antioxidante mediante la captura, inhibición y prevención de la formación de las especies reactivas de oxígeno; b) la regulación del sistema antioxidante endógeno; c) la activación de los mecanismos de reparación al contribuir en la eliminación del aducto 8-hidroxi-2'-desoxiguanosina; d) la inducción de apoptosis en células con daño al ADN; y e) la inhibición de la inflamación relacionada con su actividad antiapoptótica. Si bien, en algunos de los estudios se reportaron efectos genotóxicos, estos a su vez contribuyeron en la eliminación de células con daño genético, por lo que, no se puede considerar del todo a la actividad genotóxica de las CTV como perjudiciales para la salud(AU)

The green tea catechins (Camellia sinensis) (CTV) have beneficial effects for health associated with their antioxidant potential. Moreover, oxidative stress is one of the pathways for inducing genotoxic damage. Hence, in this review, an analysis of the antigenotoxic and genotoxic effects of CTV was carried out, emphasizing the pathways involved in these processes and their effects on health. A review of articles indexed in the PubMed® and ScienceDirect® (2021) databases with the keywords "green tea" and "green tea catechins" was carried out. Studies were delimited using the Boolean operators "AND", "OR" and "NOT" ("antigenotoxic", "genotoxic", "antioxidant" and "prooxidant"). For the most part, publications from 2016 to 2021 were considered. It was observed that the beneficial health effects of CTVs are related to: a) their antioxidant activity through the capture, inhibition and prevention of the formation of reactive oxygen species; b) the regulation of the endogenous antioxidant system; c) the activation of the repair mechanisms by contributing to the elimination of the 8-hydroxy-2'-deoxyguanosine adduct; d) the induction of apoptosis in cells with DNA damage; and e) the inhibition of inflammation related to its antiapoptotic activity. Although some of the studies reported genotoxic effects, these in turn contributed to the elimination of cells with genetic damage. Therefore, the genotoxic activity of CTV cannot be considered as harmful to health

Avaliação da citotoxicidade in vitro e genotoxicidade ex-vivo em compostos da Pavonia glazioviana Gürke (Malvaceae / Evaluation of in vitro cytotoxicity and ex-vivo genotoxicity in compounds from Pavonia glazioviana Gürke(Malvaceae)

Introdução: as terapias alternativas que utilizam plantas medicinais e fitoterápicos são bastante comuns no Brasil. Dentre várias espécies vegetais brasileiras utilizadas em terapias destacam-se as espécies da família Malvaceae. Objetivos: o presente estudo teve como objetivo avaliar a citotoxicidade in vitro e a genotoxicidade ex-vivo em compostos da Pavonia glazioviana Gürke espécie brasileira pertencente à família Malvaceae. Metodologia: métodos in vitro foram utilizados para verificar o potencial citotóxico por meio de ensaios hemolíticos e anti-hemolíticos e da análise genotóxica ex-vivo. O Extrato Etanólico Bruto (EEB) e Fração Clorofórmico (FC) foram obtidos na amostra vegetal utilizada neste estudo. Resultados: os produtos EEB-Pg e FC-Pg apresentaram baixo efeito citotóxico apenas nas concentrações de 50 e 100 µg / mL. As amostras expostas às concentrações de 500 e 1000 µg / mL apresentaram índice hemolítico alto a moderado com lise superior a 60%. Foi descrito efeito anti-hemolítico moderado em todas as amostras tratadas com 500 e 1000 µg / mL, com hemólise < 60%. Além disso, os compostos mostraram baixo efeito genotóxico ex-vivo, com um índice geral de células normais superior a 84% em todas as concentrações. Conclusões: os resultados sugerem um baixo perfil tóxico dos compostos obtidos da espécie Pavonia glazioviana, indicando limites seguros para o uso desses produtos naturais.

Introduction: alternative therapies using medicinal plants and herbal medicines are quite common in Brazil. Among several Brazilian plant species used in therapies, the species of the Malvaceae family stand out. Objetctives: the present study aimed to evaluate the in vitro cytotoxicity and ex-vivo genotoxicity in compounds of the Brazilian Pavonia glazioviana Gürke belonging to the Malvaceae family. Methodology: in vitro methods were used to verify the cytotoxic potential through hemolytic and antihemolytic assays and the ex-vivo genotoxic analysis. The Crude Etanolic Extract (CEE) and Cloroformic Fraction (CF) was obtained in vegetal sample used on this study. Results: the CEE-Pg and CF-Pg products only showed a low cytotoxic effect at the concentrations of 50 and 100 µg/mL. The exposure to the concentrations of 500 and 1000 µg/mL showed a high to moderate hemolytic index with lysis higher than 60%. A moderate anti-hemolytic effect was described in all samples treated with 500 and 1000 µg/mL, with hemolysis <60%. In addition, the compounds showed low ex-vivo genotoxic effect with a general index of normal cells greater than 84% at all concentrations. Conclusion: the results suggest a low toxic profile of the compounds obtained from the Pavonia glazioviana Gürke species belonging to the Malvaceae family, indicating safe limits for the use of these natural products.

GC-MS method for the determination of the genotoxic impurity chlorocyclohexane in trihexyphenidyl hydrochloride bulk drug / 中国药科大学学报

@#Gas chromatography-mass spectrometry (GC-MS) method was established for trace analysis of the potential genotoxic impurity chlorocyclohexane in trihexyphenidyl hydrochloride bulk drug, utilizing an RXI-5SIL MS column at isothermal temperature of 60 °C for the entire 6-minute run time.The inlet temperature was 180 °C and a split ratio of 10∶1 was used with the injection volume of 1.0 μL.The selective ion monitoring mode was set at m/z 82 for chlorocyclohexane with a detector voltage of 0.3 kV and an ion source temperature of 240 °C.The method was verified with respect to specificity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision and robustness.Good linear correlation was achieved with coefficient r of 0.999 9 in the concentration range of 59.72-493 ng/mL.The intra- and inter-day precision was satisfactory (RSD ≤ 5.0%) and robust (RSD ≤ 1.65%).The proposed method in this study can be adequately adopted as a tool for quality assurance of trihexyphenidyl hydrochloride in routine test of potential genotoxic impurity.

Determination of azide genotoxic impurity in linezolid API and glucose injection by UPLC-MS/MS / 中国药科大学学报

@#A UPLC-MS/MS method was established for the determination of the genotoxic impurity (R)-5-(azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinone in linezolid API and its glucose injection. Chromatographic separation was performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) with 0.1% formic acid water-0.1% formic acid acetonitrile (60∶40) at a flow rate of 0.3 mL/min. The UPLC-MS/MS was equipped with electrospray ionization in positive ionization mode and multiple reaction monitoring mode. The results showed that the calibration curve was linear in the range of 4-12 ng/mL and the limit of quantification was 0.073 ng/mL.The average recoveries of the low, medium and high concentration (80%,100%,120% limit concentration) loading solutions were 101.14%, 100.59% and 101.47%, respectively (RSDs:0.73%, 1.10% and 0.91%, respectively).The sample solution was stable for 6 d.No genotoxic impurity of (R)-5-(Azidomethyl)-3-[3-fluoro-4-(4-morpholinyl)phenyl]-2-oxazolidinonewas not detected in the samples of linezolid API and its glucose injection.

Covalent organic nanospheres as a fiber coating for solid-phase microextraction of genotoxic impurities followed by analysis using GC-MS / 药物分析学报

Covalent organic nanospheres(CONs)were explored as a fiber coating for solid-phase microextraction of genotoxic impurities(GTIs)from active ingredients(AIs).CONs were synthesized by an easy solution-phase procedure at 25℃.The obtained nanospheres exhibited a high specific surface area,good ther-mostability,high acid and alkali resistance,and favorable crystallinity and porosity.Two types of GTIs,alkyl halides(1-iodooctane,1-chlorobenzene,1-bromododecane,1,2-dichlorobenzene,1-bromooctane,1-chlorohexane,and 1,8-dibromooctane)and sulfonate esters(methyl p-toluenesulfonate and ethyl p-toluenesulfonate),were chosen as target molecules for assessing the performance of the coating.The prepared coating achieved high enhancement factors(5097-9799)for the selected GTIs.The strong affinity between CONs and GTIs was tentatively attributed to T-T and hydrophobicity interactions,large surface area of the CONs,and size-matching of the materials.Combined with gas chromatography-mass spectrometry(GC-MS),the established analytical method detected the GTIs in capecitabine and imatinib mesylate samples over a wide linear range(0.2-200 ng/g)with a low detection limit(0.04-2.0 ng/g),satisfactory recovery(80.03％-109.5％),and high repeatability(6.20％-14.8％)and reproducibility(6.20％-14.1％).Therefore,the CON-coated fibers are promising alternatives for the sensitive detection of GTIs in AI samples.

Determination of potential genotoxic impurities chloroacetyl chloride and chloroacetic acid in azintamide raw material / 中国药科大学学报

@#In this paper, chemical derivatization-high performance liquid chromatography was used to determine the potential genotoxic impurities chloroacetyl chloride and chloroacetic acid, respectively, in the raw material of azintamide.Derivatization was carried out using 2-nitrophenylhydrazine followed by the determination.Separation was performed on a Thermo Syncronis C18 column (250 mm × 4.6 mm, 5 μm), with mobile phase consisting of 0.1% phosphoric acid in water (A) and acetonitrile(B) by gradient elution, at a flow rate of 1 mL/min.The column temperature was 40 °C and the detection wavelength was 226 nm.The blank solvent, derivatization reagent, and azintamide did not interfere with the peak of the test substance, and the target component was well separated from the others.For impurities chloroacetyl chloride and chloroacetic acid, the limits of detection (LOD) were 7.5 ng/mL and 15 ng/mL respectively. There was a good linear relationship between the integral area and the concentration in the range of 30-300 ng/mL.The sample recovery rate was in the range of 87.37% ~ 109.75%.The two methods established in this study have good specificity, good precision, high sensitivity and simple operation, which can be used for the trace determination of potential genotoxic impurities chloroacetyl chloride and chloroacetic acid in the raw material of azintamide.

Antioxidant and antigenotoxic properties of Alpinia galanga, Curcuma amada, and Curcuma caesia

Objective: To compare the antioxidant and anti-genotoxic properties of Alpinia (A.) galanga, Curcuma (C.) amada, and C. caesia. Methods: Cytotoxicity of ethanolic extracts of A. galanga, C. amada, and C. caesia at selected doses was evaluated by trypan blue, MTT, and flow cytometry-based assays. Genotoxicity and anti-genotoxicity (against methyl methanesulfonate, 35 μM and H2O2, 250 μM) of these plants were studied by comet assay in human lymphocytes in vitro. Furthermore, DPPH, ABTS, FRAP, lipid peroxidation, and hydroxyl radical scavenging assays were performed to study the antioxidant potentials of the plants. Finally, anti-genotoxic potential of C. amada was validated in Swiss albino mice using comet assay. Phytochemical composition of C. amada was determined by GC/MS and HPLC. Results: The selected doses (2.5, 5, and 10 μg/mL) of A. galanga, C. amada, and C. caesia were non-toxic by cytotoxicity tests. All three ethanolic extracts of plant rhizomes demonstrated antioxidant and anti-genotoxic properties against methyl methanesulfonate-and H2O2-induced oxidative stress in human peripheral blood lymphocytes in vitro. Multivariate analysis revealed that various antioxidant properties of these extracts in DPPH, ABTS, and FRAP assays were strongly correlated with their total phenolic constituents. C. amada extract conferred protection against cyclophosphamide-induced DNA damage in the bone marrow cells of mice and DNA damage was significantly inhibited by 2.5 mg/kg C. amada extract. Conclusions: C. amada is rich in potentially bioactive molecules and exhibits potent antioxidant activities. Its anti-genotoxicity against cyclophosphamide-induced oxidative stress is also confirmed in this study.

Determination of bis(2-chloroethyl)amine hydrochloride and 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride in trazodone hydrochloride by UPLC-MS/MS / 药学学报

An UPLC-MS/MS method was established for the quantification of the genotoxic impurities bis(2-chloroethyl)amine hydrochloride and 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride in trazodone hydrochloride. The chromatographic separation of the two genotoxic impurities was performed on Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) at 20 ℃. A mixture of 5 mmol·L-1 ammonium hydrogen carbonate aqueous solution and acetonitrile at a flow rate of 0.3 mL·min-1 in gradient elution mode was employed as mobile phase. The UPLC-MS/MS was equipped with electrospray ionization in positive ionization mode and adopted multiple reaction monitoring mode. We found that the calibration curves of the two genotoxic impurities were linear in the range of 0.1-10 ng·mL-1. The limit of detection was 0.10 ng·mL-1 for bis(2-chloroethyl)amine hydrochloride and the average recovery was 101.53% (RSD = 4.06%). The limit of detection was 0.01 ng·mL-1 for 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride and the average recovery was 97.95% (RSD = 1.27%). The sample solution was stable for 24 h. No bis(2-chloroethyl)amine hydrochloride was detected in the samples, and the content of 1-(3-chloropropyl)-4-(3-chlorophenyl)piperazine hydrochloride in the samples was within the limit. This research provides a method to improve the quality control standards of trazadone.

Content Determination of Potential Genotoxic Impurity Maleic Hydrazide in Azintamide / 中国药房

OBJECTIVE：To establish a method for the content determination of potential genotoxic impurity maleic hydrazide in azintamide raw material. METHODS ：HPLC-FLD method was adopted. The determination was performed on Thermo Syncronis C18 column with mobile phase consisted of 0.2 mol/L acetic acid-methanol （gradient elution ）. The column temperature was set at 30 ℃，the excitation wavelength was 315 nm and emission wavelength was 389 nm. The flow rate was 1 mL/min，and the sample size was 20 μL. RESULTS：The blank solvent and azintamide did not interfere with the determination of maleic hydrazide. The linear range of maleic hydrazide was 19.5-300 ng/mL（r＝0.999 9）. The limit of detection was 4.5 ng/mL and the limit of quantification was 19.5 ng/mL. The recovery ranged from 98.79％ to 103.76％（RSDs were lower than 3.00％，n＝9）. RSDs of precision and stability （24 h）tests were no more than 5.63％，and those of durability tests were less than 2.00％（n＝6）. Maleic hydrazide was not detected in 3 batches of azinamide raw material. CONCLUSIONS ：The method is specific ，sensitive and accurate. It can be used for the trace determination of maleic hydrazide in azintamide or other matrix.

Determination of formaldehyde and glyoxal in varenicline tartrate using derivative method with HPLC / 中国药科大学学报

@#To establish a method for the determination of formaldehyde and glyoxal simultaneously in varenicline tartrate active pharmaceutical ingredient (API) and its intermediate, formaldehyde and glyoxal were derivatized by 2, 4-dinitrophenylhydrazine (2,4-DNPH) to improve the HPLC retention and UV detection sensitivity. Separation was performed on a C8 (150 mm × 4.6 mm, 5 μm) column by linear gradient elution using acetonitrile and water as the mobile phase; the detective wavelength was set at 380 nm.Formaldehyde and glyoxal were quantitatively determined by an external reference method.Linear calibration was established for both formaldehyde and glyoxal in the range from 0.094 to 1.88 μg/mL.The detection and the quantification limits were 0.047 μg/mL (19 μg/g) and 0.094 μg/mL (38 μg/g), respectively.The recoveries were (95.0±1.1)% and (99.4 ± 2.6)% for formaldehyde and glyoxal, respectively.This method has been fully validated to be applicable to quantitative analysis of trace amount of formaldehyde and glyoxal in varenicline tartrate API and its intermediate.Test results demonstrated that the contents of both formaldehyde and glyoxal met the permitted daily exposure (PDE) limits for the finished products of varenicline tartrate API as well as its intermediate, though the glyoxal contents in the crude intermediates were likely to exceed the limit.The established method is valuable for the manufacturing process and quality control of varenicline tartrate.

The improvement of micronucleus test method / 公共卫生与预防医学

Objective To simplify and optimize the micronucleus test method. Methods The preparation process of micronucleus test was simplified and optimized. In the improved method, the superfine solution was directly absorbed and discarded after cell culture, and then potassium chloride solution was added for hypotonic treatment. Then pre-fixation, centrifugation. Once the centrifugation was completed, the cells which fixed only once were directly dropped to the slide. Results The background of the slides was clear and the cells were slightly darker, but the observation of cells and micronucleus was not affected. There were a lot of binuclear cells, which can meet the counting requirements. With oil and high magnification, the image wais clearer and the background was cleaner. The cytoplasmic integrity rate, cell stain rate and the average number of cells per high magnification field of cells by the improvement method were significantly increased compared with that by the traditional methods, the probability P values were 0.0051 (χ2=7.8375), 0.0140 (χ2=6.0437) and 0.0025 (t=3.0951), respectively. The rate of micronucleus cells and cells group index had no statistical significance compared with the traditional method, the probability P values were 0.7749 (χ2=0.0817) and 0.5152 (U =0.0000), respectively. Conclusion The new method is more simple, easier to control the test quality, more reliable test results, and save time, manpower and material resources.

Efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L / In vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the rhizome of Curcuma longa L

RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.

ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.

LC-MS method development for the quantitation of potential genotoxic impurity 2-Methyl-6-nitro aniline in Telmisartan API

Current work discloses the sensitive LC-MS/MS method development for the trace level determination of genotoxicimpurity 2-Methyl-6-nitro aniline in Telmisartan. 2-Methyl-6-nitro aniline was determined by LC-MS/MS methodin selected ion monitoring mode using LiChrospher RP-18 (100 × 4.6 mm) 5.0 µm column. Gradient technique wasapplied for the elution of analytes using acetonitrile (mobile phase A) and 0.01 M ammonium acetate buffer (mobilephase B) in different ratios. The gradient program (T/%B) was set as 0/5, 2.50/15, 5.00/30, 10.00/50, 15.00/95, and20.00/95. Developed method was validated as per International Conference on Harmonization guidelines. The limit ofdetection and limit of quantitation values found for 2-Methyl-6-nitro aniline were 0.05 and 0.1 µg/ml. The developedmethod serves as an upright tool in quality control for quantitation of 2-Methyl-6-nitro aniline impurity at trace levelsin Telmisartan.

Biomonitoreo Genético en Trabajadores del Servicio de Radiología del Hospital Nacional Policial Luis N. Sáenz / Genetic Biomonitoring In Workers Of The Radiology Service Of National Police Hospital Luis N. Sáenz

Objetivo: Evaluar el efecto genotóxico en trabajadores expuestos a Rayos X en el Servicio de Radiología del Hospital Nacional Luis N. Sáenz PNP. Materiales y métodos. El tipo de estudio fue observacional, prospectivo, transversal, analítico, utilizando el ensayo cometa como técnica de análisis. La población de estudio fue de 20 trabajadores expuestos a los Rayos X y 20 personas sin exposición. Resultados. La media de longitud de migración de ADN dañado en el grupo control fue de 1,28±0.38 µm y 10,39±9.44 µm para el grupo expuesto, se compararon las medias de los grupos, obteniéndose p=0,001 significativo. El análisis de correlación para el daño de ADN, años de exposición y dosis recibida, se encontró una correlación significativa (p<0,05). Para la correlación del daño de ADN con la edad, no se encontró significancia estadística (p>0,05). Conclusiones. Los rayos X a bajas dosis permisibles pueden causar daño en la integridad del ADN, teniendo correlación con los primeros años de exposición en el personal que trabaja en el servicio radiología. Palabras clave: Genotóxico, exposición, rayos X, ensayo cometa.

Objective: To evaluate the genotoxic effect on workers exposed to X-rays in the Radiology Service of the Luis N. Sáenz National Hospital PNP. Materials and methods. The type of study was observational, prospective, cross-sectional, analytical, using the comet assay as an analysis technique. The study population was 20 workers exposed to X-rays and 20 people without exposure. Results The mean length of migration of damaged DNA in the control group was 1.28 ± 0.38 µm and 10.39 ± 9.44 µm for the exposed group, the means of the groups were compared, obtaining p = 0.001 significant. The correlation analysis for DNA damage, years of exposure and dose received, a significant correlation was found (p <0.05). For the correlation of DNA damage with age, no statistical significance was found (p> 0.05). Conclusions X-rays at low permissible doses can cause damage to DNA integrity, correlating with the first years of exposure of personnel working in the radiology service. Keywords: Genotoxic, exposure, X-ray, comet test.

Determination of N-Nitroso-N-Methyl-4-Aminobutyric Acid in Losartan Potassium Using High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry / 中国药学杂志

OBJECTIVE: To establish a method to determine the genotoxic impurity, N-nitroso-N-methyl-4-aminobytyric acid, in losartan potassium using high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-MS/MS). METHODS: The method was developed by using Shimadzu Shim-pack XR-ODS II column (2.0 mm×150 mm, 2.2 μm). Time program was conducted with mobile phase consisting of water (0.1% formic acid, A) and methanol (B). The flow rate was 0.3 mL•min-1, and the column oven temperature was maintained at 40 ℃. The samples were ionized by electrospray ionization (ESI) with multi reaction monitoring (MRM) data acquisition mode. The collision energies were -11, -13, and -13 V, CID gas was argon with pressure of 270 kPa.3 pairs of precursor, and product ions (m/z) of NMBA were 147.15→117.10, 147.15→87.10, and 147.15→44.10, respectively. RESULTS: The genotoxic impurity NMBA showed linearity between 1 and 100 ng•mL-1 with correlation coefficient of 0.999 9. The intra-day and inter-day repeatability was examined by relative standard deviations (RSDs) of retention time and peak area (RSD<1.10%, n=6 for intra-day repeatability and n=18 for inter-day repeatability). The accuracy was examined by percent recovery at three concentration levels, and the average percent recovery was between 94.40% and 98.04%. CONCLUSION: The established LC-MS/MS method is efficient for limit test and quantitation of NMBA in losartan potassium bulk drug.

Determination of genotoxic impurities of alkyl methanesulfonates in methanesulfonic acid by gas chromatography–mass spectrometry / 中国药科大学学报

@#An analytical liquid-liquid extraction-gas chromatography–mass spectrometry (LLE-GC-MS) method was established for the determination of genotoxic impurities including methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and isopropyl methanesulfonate (IMS) in methanesulfonic acid. An Agilent HP-1MS capillary column (30 m × 0.32 m, 1 μm) was used for separating the analytes by programmed heating with the inlet temperature of 220 °C. Mass spectrometry was operated in positive ion mode, and selective ion monitors were set at m/z 80 for MMS, m/z 79 for EMS, m/z 123 for IMS and m/z 56 for internal standard butyl methanesulfonate (BMS). Results showed that the baseline separation of MMS, EMS and IMS was achieved, and the blank extraction solution had no interference; good linearity was achieved in the range of 37-1 480 ng/mL for three alkyl methanesulfonates; The mean recoveries of MMS, EMS, IMS were 104.99%, 107.26%,108.85%, respectively, with RSD ≤ 4.54%. The established method has the characteristics of specific, sensitive, accurate, stable and good versatility, and has been used for the detection and control of alkyl methanesulfonate impurities in methanesulfonic acid from a variety of manufacturers.

Profiling of genotoxic impurities in a lidocaine hydrochloride injection / 中国药科大学学报

@#2,6-dimethylbenzenamine was determined as a genotoxic impurity in lidocaine hydrochloride injection, and 2-chloro-N-(2,6- dimethylphenyl) acetamide was determined as potential genotoxic impurity. An LC-MS/MS method was established to research the profiling of genotoxic impurities in active pharmaceutical ingredients (API), homemade preparation and reference preparation on column Agilent ZORBAX Eclipse Plus C18(4.6 mm250 mm，5 μm). The results show that in the homemade preparation the 2,6-dimethylbenzenamine and the 2-chloro-N-(2,6-dimethylphenyl) acetamide may be degraded under oxidation condition and alkaline condition in addition to the introduction from API preparation process. This study provides guidance for genotoxic risk assessment and prescription process optimization of lidocaine hydrochloride.

Neurotoxicity and mechanism of dental nanomaterials / 口腔疾病防治

@#Recently, the dental application of nano materials has made progress in clinical treatment, such as implant surface modification as well as antibacterial, and controlled release. However, the active physical and chemical properties of nanomaterials may pose a potential safety risk in humans. Dental nanomaterials used for oral application can be released into the blood through a variety of mechanisms, and they can penetrate the blood-brain barrier and enter the central nervous system. Moreover, nanomaterials can also directly affect the central nervous system through the olfactory nerve and via sensory nerve terminal transport, causing organic and functional damage to central nerves, and even causing neurotoxicity during embryo development. Nanomaterials can interact with biomolecules such as cells, genes, and proteins in the body, and can produce neurotoxicity through the mechanisms of inducing oxidative stress, inflammatory responses, cell autophagy, apoptosis, genotoxicity, etc. Factors affecting the toxicity of nanomaterials include particle size, concentration, and solubility. Dental nanomaterials and their pathways into the central nervous system, as well as the mechanisms that may cause neurotoxicity, will be discussed on this review.