Comprehensive two-dimensional liquid chromatography (LC×LC) can provide enhanced resolving power and higher peak capacities for the separation of complex samples. The transfer of fractions of too high eluotropic strength from the first dimension, however, can lead to peak broadening. This process is related to the column dimensions, the flow rates, mobile phase compositions, and stationary phase compatibility. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica (poly(N-isopropylacrylamide), pNIPAAm), that exhibits a change in polarity upon modest variations in column temperature. Retention is thus modulated by temperature and not by organic solvents, allowing for the use of purely aqueous mobile phases. As these aqueous mobile phases depict a very low eluotropic strength on a reversed-phase column, it facilitates band refocusing at the second-dimension column head in TRLC×RPLC. One of the remaining obstacles of TRLC is the long analysis time. In this research, the potential of this column combination in terms of analyte refocusing will be exploited. First, it is shown that upwards flow gradients can be implemented in the first dimension of TRLC×RPLC. As the flow in the second dimension is maintained at a constant level, a first-dimension flow gradient does not lead to impaired sensitivity and has no negative effects on the resulting peak size. Then, the novel combination of a downwards temperature gradient with an upwards flow gradient will be introduced to speed up the analyses further. Analysis time was, depending on the method used, reduced by 36-54%, as demonstrated by the analysis of mixtures of food additives, phenolic compounds, and small molecule pharmaceuticals mimicking impurity analysis at a 0.05% level.

Differential scanning calorimetry can be used to measure the impurity contents of pure organic substances on the principle of freezing-point depression. Impurity determination by differential scanning calorimetry with a dynamic method, which has the advantages of speediness and convenience, remains to be explored. Here, a series of acetanilide and dibenzothiophene samples with various purities was prepared through zone melting, and the samples were then analyzed by gas chromatography-mass spectrometry. A modified dynamic method, including encapsulating the analyte in a volatile pan through cold welding, remelting the analyte with a low heating rate, calculating the melted fraction considering the area of the tailing under the heat-flow curve, and reducing the error from solid-solution formation, is proposed. Encapsulating with a volatile pan using a proper torque gave an accurate result. Remelting gave a lower impurity content and a more narrow and sooth peak of heat-flow compared with the first melting. The impurity-content results calculated by the modified method were usually higher than those calculated by the ASTM standard method. For acetanilide and dibenzothiophene with impurity contents of less than 0.30%, the modified dynamic method showed good accuracy. The proposed method is applicable to determination of reference materials of organic substances with high purity owing to its accuracy and convenience.

A method for the determination of impurity components in perfluoroisobutyrile (C4F7N) gas was established by setting up a sealed pipe gas composition test system comprising an automatic six-way valve and gas chromatography-tandem mass spectrometry (GC-MS) apparatus. C4F7N commercial gases were purchased from an abroad company (AC company) and a domestic Chinese company (DC company). These gases were subjected to qualitative and quantitative analyses by our test system and separated on an Agilent GS-GASPRO column (30 m×0.32 mm×5 μm). The results showed that the C4F7N commercial gas contained N2, O2, C3HF7 and a trace amount of C3F6. In the C4F7N commercial gas from the AC company, the total amount of impurities was 0.13%, and the C4F7N content was 99.87%. In the C4F7N gas from DC Company, the total amount of impurities was 0.83%, and the C4F7N content was 99.17%. The C4F7N contents of the gases from both the companies were consistent with the claimed value (≥ 99%). The determination of the impurities C4F7N can be beneficial for the subsequent study of its insulation performance and thermal stability.

In this study, the possibilities of temperature responsive × reversed phase liquid chromatography (TRLC × RPLC) are assessed in terms of pharmaceutical impurity analysis. Due to the increased peak capacity per unit time they offer, two-dimensional LC approaches are gaining relevance for the analysis of complex drug formulations. Because the latter depicts a larger predisposition for the occurrence of an increased number of impurities, current 1D-HPLC approaches often prove insufficient. Since many LC × LC methods are limited by modulation, solvent compatibility, orthogonality, and sensitivity issues, the combination of TRLC × RPLC is explored in this work for pharmaceutical impurity analysis. As this combination of a purely aqueous separation with RPLC allows for systematic and optimization-free refocusing in the second dimension, it opens possibilities for generic LC × LC requiring minimal to no method development, in this way overcoming a major perceived contemporary hurdle of LC × LC. The approach is demonstrated with a representative mixture of 17 solutes comprising 11 corticosteroids and 6 progestogens. Orthogonality and peak capacities were assessed on three RP core-shell column selectivities (Poroshell EC-C18, phenyl-hexyl and PFP). Although the TRLC × EC-C18 combination offered somewhat better orthogonality, the combination with the PFP column proved the best for the separation at hand. Depending on the composition of the mixture, the use of full, shifted, or segmented gradients allowed facile optimization of the separation. The developed platform allowed detection of the impurities at the 0.05% level compared to a selected main compound, while also opening up possibilities for analysis of formulations comprising two active ingredients.

A micellar electrokinetic chromatography method for the determination of indomethacin impurities (4-chlorobenzoic acid, 5-methoxy-2-methyl-3-indoleacetic acid, and 3,4-dichloroindomethacin) has been developed. A 64.5/56-cm fused silica 50 μm id capillary with extended light path (150 μm id) detection window was used. Internal standard was 1-naphthylacetic acid. The analytes were separated at 30 kV with DAD detection at 224 nm. A central composite face-centered design was applied for the optimization of the separation conditions. The effect of SDS concentration, content of methanol, concentration of phosphate buffer, and pH of the buffer were studied at three levels. The optimized background electrolyte was 20 mmol/L phosphate buffer (pH 7.57) containing 58 mmol/L SDS and 0% MeOH. Sufficient resolution of all compounds with Rs ≥ 3.5 was achieved within 10 min. The method was validated for a range of 1.25-80 μg/mL of each impurity corresponding to 0.05-3.2% relative to the concentration of indomethacin (2.5 mg/mL). The calibration curves were rectilinear with correlation coefficients r2 exceeding 0.9994. The lower limit of quantification was 0.05% or 1.25 μg/mL that complies with the reporting limits regarding the ICH Q3A guideline. The method was applied to purity assay of indomethacin in both bulk drug and gel.

A fast and selective capillary electrophoresis method was developed and validated for the simultaneous determination of the hydrochloride and acetic acid content in prasugrel hydrochloride. Because of the poor chromophore, the indirect detection was chosen. Among different compositions studied as the background electrolyte, the pyromellitic acid with diethylamine (DEA) and myristyltrimethylammonium bromide (TTAB) was chosen. During the validation the specificity, linearity, accuracy, precision, range, and stability of the sample solution were confirmed. The results indicate that the method is suitable for the determination of the counter ion and impurity from the synthetic route of the pharmaceutical drug substance in the same assay.