•A derivatization HPLC method was developed to determine the residual acyl chlorides in lipophilic drug substances.•The nitro-substituted anilines and nitro-substituted phenylhydrazines were compared as the derivatization reagents.•2-nitrophenylhydrazine was selected as the suitable reagent due to its maximum UV wavelength absorbance at 395 nm.•Determination of acyl chlorides at 395 nm could largely minimize the matrix interferences from drug substances.Acyl chlorides are important acylating agents in the synthesis of active pharmaceutical ingredients. Determining the residual acyl chlorides in drug substances is a challenge due to their high reactivity and the matrix interferences from drug substances and their related impurities. This paper describes a general derivatization HPLC method for the determination of aromatic and aliphatic acyl chlorides in lipophilic drug substances. Since most drug substances have weak absorptions in the visible range (above 380 nm), the nitro-substituted anilines and nitro-substituted phenylhydrazines were selected as the derivatization reagents due to their weak basicity and red-shift of UV absorption spectra. The maximum wavelength and absorption intensity of nitro-substituted anilines decreased after derivatization with acyl chlorides, whereas the derivatization products of nitro-substituted phenylhydrazines showed the slight increases of maximum wavelength and absorbance intensity. Hence, 2-nitrophenylhydrazine was selected as the suitable derivatization reagent because the derivatives have the maximum UV wavelength absorbance at 395 nm, which could largely minimize the matrix interferences. The optimization of the concentration of 2-nitrophenylhydrazine is important for the sensitivity and stability of derivatives. Other reaction conditions including reaction temperature, time and the influence of three competitive solvents (water, methanol and ethanol) on the reaction efficiency were also studied. After derivatization with 100 μg mL−1 2-nitrophenylhydrazine at room temperature for 30 min, the method was validated for high specificity and sensitivity with the detection limits in the range of 0.01–0.03 μg mL−1. The proposed method was applied as a generic method to determine the residual acyl chlorides in lipophilic drug substances.

•A derivatization method for the residue determination of six HCAs was developed.•Different derivatization reagents were evaluated and 2-NPH was selected.•Derivatization with 2-NPH afforded selective and sensitive detection at 392 nm.•Pyridine caused dehalogenation of derivatives and was removed from our system.•The method was validated and applied for HCAs analysis in eight drug substances.A method for the determination of small halogenated carboxylic acid (HCA) residues in drug substances is urgently needed because of the potential of HCAs for genotoxicity and carcinogenicity in humans. We have now developed a simple method, involving derivatization followed by high performance liquid chromatography-diode array detection (HPLC-DAD), for the determination of six likely residual HCAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 2-bromopropionic acid and 3-chloropropionic acid) in drug substances. Different nitro-substituted phenylhydrazines (NPHs) derivatization reagents were systematically compared and evaluated. 2-Nitrophenylhydrazine hydrochloride (2-NPH·HCl) was selected as the most suitable choice since its derivatives absorb strongly at 392 nm, a region of the spectrum where most drug substances and impurities absorb very weakly. During the derivatization process, the commonly used catalyst, pyridine, caused rapid dechlorination or chlorine substitution of α-halogenated derivatives. To avoid these unwanted side reactions, a reliable derivatization method that did not use pyridine was developed. Reaction with 2-NPH·HCl using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as coupling agent in acetonitrile-water (70:30) at room temperature for 2 h gave complete reaction and avoided degradation products. The derivatives were analyzed, without any pretreatment, using gradient HPLC with detection in the near visible region. Organic acids commonly found in drug substances and other impurities did not interfere with the analysis. Good linearity (r > 0.999) and low limits of quantitation (0.05–0.12 μg mL−1) were obtained. The mean recoveries were in the range of 80–115% with RSD <5.81% except for 3-CPA in ibuprofen which was 78.5%. The intra- and inter-day precisons were expressed as RSD <1.98% and <4.39%, respectively. Finally, the proposed method was successfully used for the residue determination of the six HCAs in eight drug substance samples.

Polydiacetylene (PDA) is a promising membrane-screening tool because lipid constituents can be incorporated into the PDA framework to form lipid/PDA vesicles used as lipid bilayers. Previous reports have shown that the colorimetric signals of PDA could be utilized for the measurement of drug–lipid membrane interactions. In this study, the fluorescence signals of PDA vesicles were investigated for the measurement of lipid membrane affinity. Based on the fluorescence response of PDA vesicles (excitation wavelength 485 nm, emission wavelength 560 nm), the half maximal response concentration (EC50) was introduced for the evaluation of drug–membrane interactions. In order to validate this method, local anesthetics and flavonoids were selected as the reference compounds and their log(EC50) values correlated well with other lipid membrane affinity constants. Then the influence of buffer pH conditions and lipid constituents on the membrane affinity were investigated to show the wide application of this method using tetracaine hydrochloride as the reference compound. The particle size of vesicles before and after addition of tetracaine hydrochloride was determined to observe the extent of vesicle binding of the tested compound. The zeta potential results showed that the electrostatic interaction had less effect on the change of lipid membrane affinity at different pH value. Therefore, the hydrophobic interaction was assumed to play the most important role in the increase in lipid membrane affinity of tetracaine hydrochloride as the buffer pH value increased. The ratio of Chol in the lipid constituents affected the affinity of tetracaine hydrochloride less, but significantly weakened the sensitivity of PDA-based fluorescence signals. In summary, this work provides a simple, sensitive and reproducible PDA-based fluorescent method for the rapid measurement of lipid membrane affinity.

•ESI and APCI were evaluated for LC–MS determination of sulfonate ester PGIs.•Twelve sulfonate esters were systematically studied and APCI proved better than ESI.•Stable precursor ions [M-alkyl]− and corresponding product ions produced in APCI.•More adduct ions competed abundance in ESI which diminished the sensitivity.•Good precision and low limits of detection were obtained using APCI in SRM mode.Two ionization techniques for liquid chromatography–mass spectrometry (LC–MS) determination of sulfonate ester potentially genotoxic impurities (PGIs) were evaluated. Twelve PGIs including methyl, ethyl, propyl and isopropyl esters of methanesulfonate, benzenesulfonate and p-toluenesulfonate were studied in this research. Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources were compared in terms of performance and quality parameters for detection of the twelve PGIs. Their mass spectra obtained by APCI and ESI were very different in both fragment ions and relative abundances. In APCI negative ion mode the twelve sulfonate esters showed their stable precursor ions of [M-alkyl]−, which readily yielded product ions of [M-alkyl-CH3]− (for aliphatic sulfonate esters) or [M-alkyl-SO2]− (for aromatic sulfonate esters) with collision-induced dissociation (CID) applied; and working in selected reaction monitoring (SRM) mode has allowed limits of detection to be decreased. In the case of ESI ionization, these compounds showed their precursor ions [M+H]+, but their abundance was easily competed by formation of ions [M+NH4]+ and/or [M+Na]+, which led to poor analytical sensitivity and reproducibility. Although mobile phase additives could enhance the responses of adduct ions like [M+NH4]+ and [M+Na]+, no improvement was obtained when using SRM mode. Twelve sulfonate esters were systematically compared and APCI was shown to be a better ionization technique for rapid and sensitive determination of these PGIs. Performance of the developed approach for rapid determination of 12 PGIs was also evaluated. Quality parameters were established and good precision (relative standard deviations <8%) and very low limits (2–4 ng/mL) of detection were obtained, mainly when using APCI in SRM mode.

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug–membrane interactions occurred, membrane affinity constant Kb could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(Kb) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of Kb of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (Km) with R2 = 0.9793. For neutral compounds, the log(Kb) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (Koct) with a linear correlation coefficient R2 = 0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.