Development and evaluation of a HILIC-MS method for the determination of amino acid and non-amino acid impurities in histidine.

Developing analytical methods to assure and control the quality of amino acids has long been a challenge for food ingredient, dietary supplement, and pharmaceutical industries due to the high polarity and the absence of chromophores in many amino acids; the situation worsens further by the lack of information of impurities that could potentially be introduced during the manufacturing processes. Herein we utilize a four-step strategy including impurity identification, method development, sample analysis, and targeted impurity detection and quantitation to demystify the impurity profiles of amino acids. The effectiveness of the approach is highlighted using histidine as an example. Analysis of histidine manufacturing and degradation processes led to the identification of 12 potential impurities of histidine, including amino acids (arginine, lysine, asparagine, aspartic acid, alanine, and glycine) and non-amino acid impurities (histamine, histidinol, 4-imidazoleacrylic acid, 4-imidazoleacetic acid, ß-imidazolelactic acid, and urea). A HILIC method using Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm) and a mobile phase system consisting of ammonium formate buffer at pH 3.2 in water and 0.1% formic acid in acetonitrile coupled with a single quadrupole mass spectrometer was developed for the detection and quantitation of the proposed impurities. Evaluation of 11 commercial histidine samples using the developed method revealed distinct impurity profiles, as a fingerprint for each sample; seven of the 12 proposed impurities were detected in histidine samples tested. The developed method was evaluated in terms of specificity, linearity, range, accuracy, precision, and sensitivity (LOQ: 2.5-60.6 ng/mL) for its suitability for compendial applications. Given the high degree of overlap between the proposed and the detected impurities, the approach could be utilized to strengthen USP standards for controlling the quality of histidine. Extension of the strategy to the analysis of other amino acids is currently underway.

Pharmaceutical analysis by NMR can accommodate strict impurity thresholds: The case of choline.

The ICH guidelines recommend reporting thresholds for regular impurities in drug substances at the level of 0.05% or 0.03% (w/w) depending on the maximum daily intake. Therefore, any instrumental method of analysis applicable to the impurity analysis should be able to detect and quantify the analytes at those levels. This investigation was designed to verify the suitability of 1H NMR spectroscopy for the detection of impurities, as a first step in the process before attempting quantification. In order to minimize demand on equipment, this study employed a 400 MHz instrument for structural confirmation and signal assignments of choline (1) and O-(2-hydroxyethyl)choline (2), a known impurity. The limit of detection (LOD) of 2 in 10 mg of 1 was established as 0.01% on a 400 MHz instrument and 2% on a 60 MHz (benchtop) NMR spectrometer. Thus, impurities for which quantification is required are readily detected at 400 MHz or above. These results are in contrast to the widespread belief that 1H NMR sensitivity is insufficient for pharmaceutical impurity analysis. The choice of solvent was recognized as a critical parameter for 1H NMR LOD analysis. Furthermore, publicly available NMR raw data (HMDB) proved to be valuable for unveiling the otherwise cryptic information hidden in complex signal patterns via 1H NMR iterative Full Spin Analysis. Finally, the study uncovered the less noticed, yet characteristic, 14N-1H coupling in the -N+(CH3)3 groups, adding strong arguments for the Raw NMR Data Initiative. Collectively, the data prove that the analytical capabilities of high-field NMR easily fulfill the ICH requirements for detection of impurity in the presence of an actual substance of interest which makes it a step closer to achieving regulatory standards.

Rapid detection of urushiol allergens of Toxicodendron genus using leaf spray mass spectrometry.

A new ambient ionization method--leaf-spray mass spectrometry--is used to detect allergenic urushiols directly from poison ivy (T. radicans) leaves with no sample preparation. These simple measurements show all the urushiols previously reported using liquid chromatography mass spectrometry methods. Tandem mass spectrometry analysis of the leaf spray ions confirms the identifications. Enhanced detection of some urushiols was achieved in the negative mode with the addition of chloride anions to the spray solvent.

In situ analysis of agrochemical residues on fruit using ambient ionization on a handheld mass spectrometer.

We describe a rapid in situ method for detecting agrochemicals on the surface or in the tissue of fruit using a portable mass spectrometer equipped with an ambient ionization source. Two such ionization methods, low temperature plasma (LTP) and paper spray (PS), were employed in experiments performed at a local grocery store. LTP was used to detect diphenylamine (DPA) directly from the skin of apples in the store and those treated after harvest with DPA were recognized by MS and MS/MS. These data therefore allowed ready distinction between organic and non-organic apples. DPA was also found within the internal tissue of purchased apples and its distribution was mapped using LTP. Similarly, thiabendazole residues were detected on the skin of treated oranges in a grocery store experiment in which paper spray was performed by wiping the orange surface with a moist commercial lens wipe and then applying a high voltage to ionize the chemicals directly from the wipe. The handheld mass spectrometer used in these measurements is capable of performing several stages of tandem mass spectrometry (up to MS(5)); the compounds on the fruit were identified by their MS/MS fragmentation patterns. Protonated DPA (m/z 170) produced a characteristic MS(2) fragment ion at m/z 92, while thiabendazole was identified by MS(3) using precursor to fragment ion transitions m/z 202 →m/z 175 →m/z 131. These particular examples exemplify the power of in situ analysis of complex samples using ambient ionization and handheld mass spectrometers.

Evaluation of a differential mobility spectrometer/miniature mass spectrometer system.

A planar differential mobility spectrometer (DMS) was coupled to a Mini 10 handheld rectilinear ion trap (RIT) mass spectrometer (MS) (total weight 10 kg), and the performance of the instrument was evaluated using illicit drug analysis. Coupling of DMS (which requires a continuous flow of drift gas) with a miniature MS (which operates best using sample introduction via a discontinuous atmospheric pressure interface, DAPI), was achieved with auxiliary pumping using a 5 L/min miniature diaphragm sample pump placed between the two devices. On-line ion mobility filtering showed to be advantageous in reducing the background chemical noise in the analysis of the psychotropic drug diazepam in urine using nanoelectrospray ionization. The combination of a miniature mass spectrometer with simple and rapid gas-phase ion separation by DMS allowed the characteristic fragmentation pattern of diazepam to be distinguished in a simple urine extract at lower limits of detection (50 ng/mL) than that achieved without DMS (200 ng/mL). The additional separation power of DMS facilitated the identification of two drugs of similar molecular weight, morphine (average MW = 285.34) and diazepam (average MW = 284.70), using a miniature mass spectrometer capable of unit resolution. The similarity in the proton affinities of these two compounds resulted in some cross-interference in the MS data due to facile ionization of the neutral form of the compound even when the ionic form had been separated by DMS.

A study of the performance of an ion shutter for drift tubes in atmospheric pressure ion mobility spectrometry: computer models and experimental findings.

Ion mobility spectra are initiated when ions, derived from a sample, are pulsed or injected through ion shutters into a drift region. The effect on signal intensity from electric fields arising from the shutter grids (E(s)) and a superimposed electric field of the drift tube (E(d)) was determined experimentally and simulated computationally for ion motion at ambient pressure. The combination of these two fields influenced shutter performance in three ways: (1) intensity of an ion peak was suppressed by increased current in the baseline due to continuous leakage of ions into the drift region from insufficient E(s) to block ion motion when needed, at a given value of E(d); (2) the ion shutter provided maximum peak intensity with some optimal ratio of E(s)/E(d) when ions were fully blocked except using the injection time; (c) the signal intensity was reduced when the blocking voltage of the ion shutter exceeded this optimal E(s)/E(d) ratio from ion depletion at the shutter grids. The optimal ratio from the computer models was equal to 1.50, whereas a value of 2.50 was obtained from the experimental findings. This difference was attributed to nonideal geometry with the grids of the shutter and the conducting elements in the drift tube establishing both E(s) and E(d). As both the experimental and modeling results demonstrated, a mobility dependence of ion yield from the ionization source was found to cause a mobility dependent ion signal at the collector electrode.