Highly sensitive two-dimensional ion chromatography mass spectrometry method for nitrite determination in hydroxypropyl methylcellulose.

Due to their potential adverse health effects, some N-nitrosamines in drug products are strictly regulated with very low maximum daily intake limits. Nitrosamines can be formed from the reaction of nitrite and secondary or tertiary amines when both species co-exist in the drug synthesis or formulation process. One key strategy to mitigate nitrosamine risk in drugs is to select low-nitrite containing pharma excipients for formulation. It is necessary to develop a sensitive method for trace nitrite determination in pharma excipients as it enables drug producers to study nitrosamine formation kinetics and select excipient suppliers. This study details the development and validation of a two-dimensional ion chromatography mass spectrometry (2D-IC/MS) method for trace nitrite determination in hydroxypropyl methylcellulose (HPMC), one of the most important pharmaceutical excipients used in many drug formulations. The 2D-IC system was operated in heart-cutting mode with a concentrator column coupling the two dimensions. A standard bore anion-exchange column was used in the first dimension (1D) to enable a large volume injection for increased sensitivity and provide improved resolution between nitrite and the interfering chloride peak. A high efficiency microbore anion-exchange column with different selectivity was used in the second dimension (2D) to resolve nitrite from other interfering species. The use of 2D-IC resulted in significantly improved resolution, solving the sensitivity loss issue due to ion suppression from an otherwise 1D separation. MS detection with selective ion monitoring and isotope labeled nitrite internal standard further improve the method specificity, accuracy, and ruggedness, as compared with conductivity detection. For trace determination, it is also extremely important to have a clean blank. For this purpose, a novel cleaning procedure using a strong anion wash was developed to remove nitrite contamination from labware. The optimized method was validated with linearity of nitrite in the concentration range of 18.5-5005.8 ng/g having a regression coefficient of >0.9999, precision with RSD at 3.5-10.1 % and recovery of 90.5-102.4 %. The limit of detection and limit of quantitation were 8.9 and 29.6 ng/g relative to the HPMC sample, or equivalent to 89 and 296 pg/g in the sample solution, respectively.

Minimize Precolumn Band Broadening with Immiscible Solvent Sandwich Injection.

We investigated the possibility of reducing the effect of precolumn band broadening (PreCBB) by sandwiching the sample between two small plugs of an immiscible liquid. It has been found that in cases of severe PreCBB, improvements in peak efficiency can amount up to 20 times for the early-eluting compounds. For smaller degrees of PreCBB, the gain on the efficiency of early-eluting compounds is smaller (order of 50%), yet it is still significant. It has been verified that the presence of the immiscible fluid sandwich does not affect the repeatability of the analysis nor the linearity of the calibration curves used for analyte quantitation. It is also shown that the main effect of the two sandwich plugs is the minimization of the dispersion in the precolumn transfer tubing itself, which makes the method fundamentally different from pure on-column focusing methods such as the performance optimizing injection sequence (POISe) method. It is further demonstrated that both halves of the sandwich are needed, since the beneficial effect is clearly much smaller when only one plug is present. A drawback of the method is that some of the late-eluting peaks are slightly adversely affected by the presence of the sandwich liquid in the case where 127 µm i.d. tubing was used. The mechanism for this peak deterioration effect is at present still unclear but only occurs under gradient conditions and is clearly linked to the size of the sandwich plugs (the smaller the plugs, the smaller the adverse effect) and the internal diameter of the tubing used between the injection valve and the column.

Parts per billion of nitrite in microcrystalline cellulose by ion chromatography mass spectrometry with isotope labeled internal standard.

Regulatory authorities like the U.S. Food and Drug Administration (FDA) have set strict specification levels for N-nitrosamines in finished drug products. Nitrite is a potential precursor for the formation of probable carcinogenic N-nitrosamines when secondary or tertiary amines are also present in the active pharmaceutical ingredient (API) synthesis or drug formulation process. An accurate and sensitive determination of nitrite will be useful when a drug product manufacturer chooses to investigate the reaction kinetics between nitrite and amines or to select appropriate excipients for its drug formulation. Pharmaceutical excipient manufacturers may also need an accurate nitrite measurement to investigate the nitrite content in their excipients. This study details the development and validation of an ion chromatography mass spectrometry (IC-MS) method for trace nitrite determination in microcrystalline cellulose materials, one of the important pharmaceutical excipients used in many drug formulations. MS operated under selected ion monitoring mode was used to solve the commonly encountered interference issue with conductivity detection, and nitrite isotope internal standard was employed to address the ion suppression issue with MS detection. The installation of an after-column "jumper" to flush water with an auxiliary pump through the MS when it is not used for data collection avoided sensitivity loss due to trace salt accumulation in the ion source. Validation of the optimized method was satisfactory, with linearity of nitrite in the concentration range of 0.02-7.50 ppm (µg/g) having a regression coefficient of > 0.999, precision of RSD < 9.5% at 0.03 ppm and RSD < 3.4% at 0.4 ppm and recovery of 92.0-103.0%. The limit of detection and limit of quantitation were 0.005 and 0.016 ppm, respectively.

Maximizing two-dimensional liquid chromatography peak capacity for the separation of complex industrial samples.

We report on a study on the use of 2D-LC in industry wherein we maximized the peak capacity by serially coupling up to six 15 cm long columns in the 1st dimension. For the considered aromatic amine oligomer sample, the combination of reverse phase pentafluorophenyl column providing selectivity based on π-π interaction in the 1st and a more retaining reverse phase polymeric C18 column in the 2nd dimension proved to give the highest orthogonality, calculated to be 0.82. Whereas a 1D run on a single column revealed around 120 compounds, the optimized 2D-LC system revealed around 940 compounds. To achieve this, flow splitting to improve the peak capacity in the 1st dimension and shifting gradients in the 2nd dimension were used. The overall peak capacity of the system was calculated to be 11,000 with correction for orthogonality and undersampling. The total analysis time with the 6-column system was around 20 h.

Elimination of N,O-bis(trimethylsilyl)trifluoroacetamide interference by base treatment in derivatization gas chromatography mass spectrometry determination of parts per billion of alcohols in a food additive.

A novel base treatment followed by liquid-liquid extraction was developed to remove the interference of excess derivatization reagent BSTFA [N,O-Bis(trimethylsilyl)trifluoroacetamide] and its byproducts for trace determination of 1-chloro-2-propanol and 2-chloro-1-propanol in a food additive. The corresponding trimethylsilyl derivatives were analyzed by gas chromatography mass spectrometry (GC/MS) detection in selective ion monitoring mode. Due to a large volume splitless injection needed for achieving the required sensitivity, excess BSTFA in the derivatization sample solution interfered with the trimethylsilyl derivatives of the analytes of interest, making their quantitation not attainable. Efforts were made to decompose BSTFA while keeping the trimethylsilyl derivatives intact. Water or aqueous sulfuric acid treatment converted BSTFA into mainly N-trimethylsilyltrifluoroacetamide, which partitions between aqueous and organic layers. In contrast, aqueous sodium hydroxide decomposed BSTFA into trifluoroacetic acid, which went entirely into the aqueous layer. No BSTFA or its byproduct N-trimethylsilyltrifluoroacetamide or trifluroacetamide was found in the organic layer where the derivatized alcohols existed, which in turn completely eliminated their interference, enabling accurate and precise determination of parts per billion of the short-chain alcohols in the food additive. Contrary to the conventional wisdom that a trimethylsilyl derivative is susceptible to hydrolysis, the derivatized short-chain alcohols were found stable even in the presence of 0.17N aqueous sodium hydroxide as the improved GC/MS method was validated successfully, with a satisfactory linearity response in the concentration range of 10-400ng/g (regression coefficient greater than 0.999), good method precision (<4%), good recovery (90-98%), and excellent limit of detection (3ng/g) and limit of quantitation (10ng/g).

Stereoselective cascade reactions that incorporate a 7-exo acyl radical cyclization.

[reaction: see text] Radical cascades that feature a 7-exo acyl radical cyclization followed by a 6-exo or 5-exo alkyl radical cyclization proceed with very good yields and diastereoselectivities. Two stereocenters are created by the reaction, and a single isomeric product was obtained from each of the five substrates examined. The relative configurations of the products are consistent with cyclizations occurring via chairlike or pseudochairlike transition states.