An update on the current status and prospects of nitrosation pathways and possible root causes of nitrosamine formation in various pharmaceuticals.

Over the last two years, global regulatory authorities have raised safety concerns on nitrosamine contamination in several drug classes, including angiotensin II receptor antagonists, histamine-2 receptor antagonists, antimicrobial agents, and antidiabetic drugs. To avoid carcinogenic and mutagenic effects in patients relying on these medications, authorities have established specific guidelines in risk assessment scenarios and proposed control limits for nitrosamine impurities in pharmaceuticals. In this review, nitrosation pathways and possible root causes of nitrosamine formation in pharmaceuticals are discussed. The control limits of nitrosamine impurities in pharmaceuticals proposed by national regulatory authorities are presented. Additionally, a practical and science-based strategy for implementing the well-established control limits is notably reviewed in terms of an alternative approach for drug product N-nitrosamines without published AI information from animal carcinogenicity testing. Finally, a novel risk evaluation strategy for predicting and investigating the possible nitrosation of amine precursors and amine pharmaceuticals as powerful prevention of nitrosamine contamination is addressed.

A Stability-Indicating Assay for Tetrahydrocurcumin-Diglutaric Acid and Its Applications to Evaluate Bioaccessibility in an In Vitro Digestive Model.

A simple and reliable ultra-high-performance liquid chromatographic (UHPLC) method was developed and validated for determination of tetrahydrocurcumin diglutaric acid (TDG) and applied for evaluation of its bioaccessibility. The analytical method was validated to demonstrate as a stability-indicating assay (SIA) according to the ICH Q2(R1) guidelines under various force degradation conditions including thermal degradation, moisture, acid and base hydrolysis, oxidation, and photolysis. The developed chromatographic condition could completely separate all degradants from the analyte of interest. The method linearity was verified in the range of 0.4-12 µg/mL with the coefficient of determination (r2) > 0.995. The accuracy and precision of the method provided %recovery in the range of 98.9-104.2% and %RSD lower than 4.97%, respectively. The limit of detection and quantitation were found to be 0.25 µg/mL and 0.40 µg/mL, respectively. This method has been successfully applied for the bioaccessibility assessment of TDG with the bioaccessibility of TDG approximately four fold greater than THC in simulated gastrointestinal fluid. The validated SIA method can also benefit the quality control of TDG raw materials in pharmaceutical and nutraceutical development.

Current status and prospects of development of analytical methods for determining nitrosamine and N-nitroso impurities in pharmaceuticals.

Nitrosamine impurities in pharmaceuticals have recently been concerned for several national regulatory agencies to avoid carcinogenic and mutagenic effects in patients. The demand for highly sensitive and specific analytical methods with LOQs in the ppb and sub-ppb ranges is among the most significant challenges facing analytical scientists. In addition, artifactual nitrosamine formation during sample preparation and injection leading to overestimation of nitrosamines has received considerable attention. Numerous analytical methodologies have been reported for quantifying nitrosamine impurities in active pharmaceutical ingredients and medicinal products at the interim limit criteria as preventive measures. In this review, we meticulously discuss those reported gas and liquid chromatographic methods for nitrosamine determination in pharmaceuticals in aspects of chromatographic conditions and sensitivity of detection. We also introduce the potential of novel fluorescence-based methods recently developed to rapidly screen nitrosamine impurities. In addition, the review assesses the nitrosation assay procedure (NAP test), which is expected to be a future preventive measure for screening potential nitrosation and identifying suspected contamination with N-nitroso or other potential mutagenic impurities during the drug development process.

Development of a Sensitive Headspace Gas Chromatography-Mass Spectrometry Method for the Simultaneous Determination of Nitrosamines in Losartan Active Pharmaceutical Ingredients.

Nitrosamine impurities in angiotensin II receptor antagonists (sartans) containing a tetrazole group represent an urgent concern for active pharmaceutical ingredient (API) manufacturers and global regulators. Regarding safety, API manufacturers must develop methods to monitor the levels of each nitrosamine impurity before individual batch release. In this study, we developed and validated a sensitive, selective, and high-throughput method based on headspace gas chromatography-mass spectrometry (HS-GC-MS) for the simultaneous determination of four nitrosamines in losartan potassium API with simple sample preparation. N-Nitrosodimethylamine (NDMA, m/z 74), N-nitrosodiethylamine (NDEA, m/z 102), N-nitrosoethylisopropylamine (EIPNA, m/z 116), and N-nitrosodiisopropylamine (DIPNA, m/z 130) levels were quantified using an electron impact, single quadrupole mass spectrometer under a selected-ion-monitoring acquisition method. The method was validated according to the Q2(R1) ICH guidelines. The calibration curves of the assay ranged from 25 to 5000 ng/mL with limits of quantitation of 25 ppb for NDMA and NDEA and 50 ppb for DIPNA and EIPNA. The accuracy of the developed method ranged from -7.04% to 7.25%, and the precision %CV was ≤11.5. Other validation parameters, including specificity, stability, carryover, and robustness, met the validation criteria. In conclusion, the developed method was successfully applied for the determination of nitrosamines in losartan potassium APIs.

A stability-indicating UPLC method for the determination of curcumin diethyl disuccinate, an ester prodrug of curcumin, in raw materials.

A stability-indicating reversed-phase ultra-performance liquid chromatographic (UPLC) method for quantitative analysis of curcumin diethyl disuccinate (CDD) in raw materials was developed for applications in product development and quality control. Chromatographic separation was performed using the Waters ACQUITY UPLC® H-Class system consisting of an ACQUITY UPLC® BEH C18 (1.7 µm, 2.1 × 50 mm) column and a photodiode array detector set at a wavelength of 400 nm. The mobile phase consisting of 2%v/v acetic acid in water and acetonitrile was delivered at a flow rate of 0.3 mL/min under gradient elution program. The method was validated according to the ICH Q2(R1) guideline for the validation of analytical procedures. The method was found to be linear over the concentration range of 8-12 µg/mL with the coefficient of determination >0.995. The accuracy of the method established as %recovery ranged from 98.3 - 100.8%. The precision of the method expressed as %CV was found to be <1%. The coelution of degradation products from six stress test conditions was not observed. The method was robust under the variation of chromatographic parameters. The method was successfully applied in the determination of CDD content in raw materials.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Simple Analysis of Sumatriptan and its Application in Bioequivalence Study.

This work demonstrated a sensitive, selective, and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of sumatriptan in human plasma samples. Terazosin was used as an internal standard to minimize the variability during sample processing and detection. Sample cleanup prior to chromatographic analysis was accomplished by liquid-liquid extraction (LLE) with tert-butyl methyl ether (t-BME). The separation was performed on a reversed-phase Symmetry® C18 column (150 × 4.6 mm i.d., 5 µm) under a gradient mode, using a 0.2% formic acid aqueous solution and acetonitrile at a flow rate of 0.5 mL/min. Sumatriptan (m/z 296.26→251.05) and terazosin (m/z 388.10→290.25) were quantified using a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) under the positive ion mode. The method was fully validated following US-FDA and EMA guidelines. The LC-MS/MS assay had a calibration range of 0.5-50.0 ng/mL. The assay was precise and accurate with a between-run precision of <9.51%, and between-run accuracy between -7.27 to 8.30%. The developed method was subsequently applied in the determination of plasma concentration-time profile of a sumatriptan 50-mg tablet following oral administration in healthy volunteers.

Scale-Up Synthesis and In Vivo Anti-Tumor Activity of Curcumin Diethyl Disuccinate, an Ester Prodrug of Curcumin, in HepG2-Xenograft Mice.

Previously, we synthesized curcumin and a succinate ester prodrug of curcumin namely curcumin diethyl disuccinate (CurDD) in the lab scale, which yielded hundred milligrams to few grams of the compounds. CurDD was found to be more stable in a phosphate buffer pH 7.4 and exhibited better cytotoxicity against Caco-2 cells than curcumin. Here, the one-pot syntheses of curcumin and CurDD were scaled up to afford multigram quantities of both compounds for preclinical studies using a 10-L chemical reactor. The key steps for the synthesis of curcumin were the formation of boron-acetylacetone complex and the decomplexation of boron-curcumin complex. The synthesis of CurDD could be achieved via a one-step esterification between curcumin and succinic acid monoethyl ester chloride using 4-(N,N-dimethylamino)pyridine as a catalyst. The synthesized curcumin and CurDD were then investigated and compared for an anti-tumor activity in HepG2-xenograft mice. CurDD could reduce the tumor growth in HepG2-xenograft mice better than curcumin. CurDD also exerted the stronger inhibition on VEGF secretion, COX-2 and Bcl-2 expression and induced higher Bax expression in comparison with curcumin. The results suggest that CurDD is a promising prodrug of curcumin and has a potential to be further developed as a therapeutic agent or an adjuvant for the treatment of hepatocellular carcinoma.

A curcumin-diglutaric acid conjugated prodrug with improved water solubility and antinociceptive properties compared to curcumin.

In this work, a curcumin-diglutaric acid (CurDG) prodrug was synthesized by conjugation of curcumin with glutaric acid via an ester linkage. The water solubility, partition coefficient, release characteristics, and antinociceptive activity of CurDG were compared to those of curcumin. The aqueous solubility of CurDG (7.48 µg/mL) is significantly greater than that of curcumin (0.068 µg/mL). A study in human plasma showed that the CurDG completely releases curcumin within 2 h, suggesting the ability of CurDG to serve as a prodrug of curcumin. A hot plate test in mice showed the highest antinociceptive effect dose of curcumin at 200 mg/kg p.o., whereas CurDG showed the same effect at an effective dose of 100 mg/kg p.o., indicating that CurDG significantly enhanced the antinociceptive effect compared to curcumin. The enhanced antinociceptive effect of CurDG may be due to improved water solubility and increased oral bioavailability compared to curcumin.

Determination of levocetirizine in human plasma by LC-MS-MS: validation and application in a pharmacokinetic study.

A fast and simple sample cleanup approach for levocetirizine in human was developed using protein precipitation coupled with LC-MS-MS. Samples were treated with 6% trichloroacetic acid in water prior to LC-MS-MS analysis. Chromatographic separation was performed on a reverse phase column with an isocratic mobile phase of acetonitrile and 10 mM ammonium formate pH 3.5 (80:20, v/v) at a flow rate of 1.0 mL/min. The run time was 3.5 min. Mass parameters were optimized to monitor transitions at m/z [M+H](+) 389.0→201.0 for levocetirizine and m/z [M+H](+) 375.3→201.0 for hydroxyzine as internal standard. The lower limit of quantification and the dynamic range were 1.00 and 1.00-500 ng/mL, respectively. Linearity was good for intraday and interday validations (r(2) ≥ 0.995). The mean recoveries were 59 and 69% for levocetirizine and hydroxyzine, respectively. Matrix effect was acceptable with %CV < 15. Hemolytic effect was negligible. Levocetirizine was stable in human plasma for 27 h at room temperature (25°C), for 16 weeks frozen at -70°C, 4 weeks frozen at -20°C, for 24 h in an autosampler at 15°C and for three freeze/thaw cycles. The validated method was applied in a pharmacokinetic study to determine the concentration of levocetirizine in plasma samples. The study provides a fast and simple bioanalytical method for routine analysis and may be particularly useful for bioequivalence studies.

Time-dependent slowly-reversible inhibition of monoamine oxidase A by N-substituted 1,2,3,6-tetrahydropyridines.

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.

Effects of different carboxylic ester spacers on chemical stability, release characteristics, and anticancer activity of mono-PEGylated curcumin conjugates.

We investigated the effects of different carboxylic ester spacers of mono-PEGylated curcumin conjugates on chemical stability, release characteristics, and anticancer activity. Three novel conjugates were synthesized with succinic acid, glutaric acid, and methylcarboxylic acid as the respective spacers between curcumin and monomethoxy polyethylene glycol of molecular weight 2000 (mPEG(2000) ): mPEG(2000) -succinyl-curcumin (PSC), mPEG(2000) -glutaryl-curcumin (PGC), and mPEG(2000) -methylcarboxyl-curcumin (PMC), respectively. Hydrolysis of all conjugates in buffer and human plasma followed pseudo first-order kinetics. In phosphate buffer, the overall degradation rate constant and half-life values indicated an order of stability of PGC > PSC > PMC > curcumin. In human plasma, more than 90% of curcumin was released from the esters after incubation for 0.25, 1.5, and 2 h, respectively. All conjugates exhibited cytotoxicity against four human cancer cell lines: Caco-2 (colon), KB (oral cavity), MCF7 (breast), and NCI-H187 (lung) with half maximal inhibitory concentration (IC(50) ) values in the range of 1-6 µM, similar to that observed for curcumin itself. Our results suggest that mono-PEGylation of curcumin produces prodrugs that are stable in buffer at physiological pH, release curcumin readily in human plasma, and show anticancer activity.

Synthesis, characterization and biological evaluation of succinate prodrugs of curcuminoids for colon cancer treatment.

A novel series of succinyl derivatives of three curcuminoids were synthesized as potential prodrugs. Symmetrical (curcumin and bisdesmethoxycurcumin) and unsymmetrical (desmethoxycurcumin) curcuminoids were prepared through aldol condensation of 2,4-pentanedione with different benzaldehydes. Esterification of these compounds with a methyl or ethyl ester of succinyl chloride gave the corresponding succinate prodrugs in excellent yields. Anticolon cancer activity of the compounds was evaluated using Caco-2 cells. The succinate prodrugs had IC50 values in the 1.8-9.6 µM range, compared to IC50 values of 3.3-4.9 µM for the parent compounds. Curcumin diethyl disuccinate exhibited the highest potency and was chosen for stability studies. Hydrolysis of this compound in phosphate buffer at pH 7.4 and in human plasma followed pseudo first-order kinetics. In phosphate buffer, the k(obs) and t(½) for hydrolysis indicated that the compound was much more stable than curcumin. In human plasma, this compound was able to release curcumin, therefore our results suggest that succinate prodrugs of curcuminoids are stable in phosphate buffer, release the parent curcumin derivatives readily in human plasma, and show anti-colon cancer activity.

Identification of isobaric product ions in electrospray ionization mass spectra of fentanyl using multistage mass spectrometry and deuterium labeling.

Isobaric product ions cannot be differentiated by exact mass determinations, although in some cases deuterium labeling can provide useful structural information for identifying isobaric ions. Proposed fragmentation pathways of fentanyl were investigated by electrospray ionization ion trap mass spectrometry coupled with deuterium labeling experiments and spectra of regiospecific deuterium labeled analogs. The major product ion of fentanyl under tandem mass spectrometry (MS/MS) conditions (m/z 188) was accounted for by a neutral loss of N-phenylpropanamide. 1-(2-Phenylethyl)-1,2,3,6-tetrahydropyridine (1) was proposed as the structure of the product ion. However, further fragmentation (MS(3)) of the fentanyl m/z 188 ion gave product ions that were different from the product ion in the MS/MS fragmentation of synthesized 1, suggesting that the m/z 188 product ion from fentanyl includes an isobaric structure different from the structure of 1. MS/MS fragmentation of fentanyl in deuterium oxide moved one of the isobars to 1 Da higher mass, and left the other isobar unchanged in mass. Multistage mass spectral data from deuterium-labeled proposed isobaric structures provided support for two fragmentation pathways. The results illustrate the utility of multistage mass spectrometry and deuterium labeling in structural assignment of isobaric product ions.

A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts.

INTRODUCTION: Turmeric (Curcuma longa) extracts contain three curcuminoids (curcumin, desmethoxycurcumin and bisdesmethoxycurcumin) as major bioactive substances. Previously reported HPLC-UV methods for the determination of curcuminoids have several disadvantages, including unsatisfactory separation times, poor resolution and/or complicated solvent mixtures with gradient elution. OBJECTIVE: To develop a simple isocratic HPLC-UV method for the simultaneous determination of individual curcuminoids for the quality control of turmeric extracts. METHODOLOGY: The sample was prepared by dissolving the extract in acetonitrile and subsequently diluting with 50% acetonitrile. This solution was analysed by reverse-phase chromatography on an Alltima C(18) column with isocratic elution of acetonitrile and 2% v/v acetic acid (40:60, v/v) at a flow rate of 2.0 mL/min, a column temperature of 33 degrees C, and UV detection at 425 nm. The method was validated and applied for quantification of individual curcuminoids in commercial turmeric extracts. RESULTS: The method allowed simultaneous determination of curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in the concentration ranges of 10-60, 4-24 and 0.5-3.0 microg/mL, respectively. The limits of detection and quantification were, respectively, 0.90 and 2.73 microg/mL for curcumin, 0.84 and 2.53 microg/mL for desmethoxycurcumin and 0.08 and 0.23 microg/mL for bisdesmethoxycurcumin, and the percentage recoveries were, respectively, 99.16-101.75 (%RSD < or = 1.11%), 99.50-101.01 (%RSD < or = 1.74%) and 99.67-101.92 (RSD < or = 1.31%). CONCLUSION: The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of turmeric extracts containing the three curcuminoid compounds as the main principles in the herb.

Simple HPLC determination of benzalkonium chloride in ophthalmic formulations containing antazoline and tetrahydrozoline.

A simple and rapid analytical procedure for routine quantification of n-C12H25 and n-C14H29 benzalkonium chloride (C-12 and C-14 BKC) homologs in ophthalmic formulations containing antazoline HCl and tetrahydrozoline HCl by high-performance liquid chromatography was developed and validated. The ophthalmic solution samples can be directly analyzed by reversed-phase on HiQ-Sil C18 column (4.6 mm x 150 mm, i.d., 5-microm particle size) with acetonitrile-sodium acetate buffer (pH 5.0; 0.2 M) (70:30, v/v) as mobile phase. UV Detection was carried out at 262 nm. The method was linear over the selected concentration and ranged from 0.03 to 0.10 mg/ml (r2 = 0.9999) and from 0.01 to 0.05 mg/ml (r2 = 0.9979) for C-12 and C-14 BKC homologs, respectively. The mean percent recoveries were 100.2 and 102.6 and the percent CV values were 1.3 and 3.5 for C-12 and C-14 BKC homologs, respectively. The results demonstrated the good linearity, accuracy, and precision. The method was applied to determine two commercial ophthalmic formulations, and the percent label amounts of total BKC contents were found to be 99.7 and 103.2.