Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH 8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine ß-lactoglobulin A.

Variation in the Content of Bioactive Compounds in Infusions Prepared from Different Parts of Wild Polish Stinging Nettle (Urtica dioica L.).

Nettle is a common plant that offers many health benefits and is grown all over the world. The content of active compounds in roots, stems, and leaves was determined based on the extraction procedure optimized using the Central Composite Design. Flavonols, phenolic acids, trigonelline, nicotinamide, nicotinic acids, and short-chain organic acids were determined with the use of LC-MS/MS and capillary isotachophoresis. Trigonelline, which was not previously reported in the roots and stems of nettle, was found in all parts of the plant and considerable variations in its content were observed (2.8-108 µg g-1). Furthermore, the Principal Component Analysis taking into account more variables demonstrated differences in the content of bioactive components between roots and aerial parts of nettle.

Comparison of 1D a 2D ITP-MS performance parameters and application possibilities: Ultratrace determination of B vitamins in human urine.

The possibility to investigate analytes at ultra-low concentration levels still remains a hot topic in bioanalysis. In this area, various preconcentration techniques are an integral part of analytical procedures. When applying electromigration separation techniques, an isotachophoresis has been advantageously employed many times for this purpose. To solve current biomedical tasks effectively, an advanced two-dimensional isotachophoretic instrument (in a hydrodynamically closed separation system with an enhanced sample load capacity) hyphenated with mass spectrometry (ITP-ITP-MS) has been proposed by Foret and coworkers. As a continuation, this work represents the first study dealing with a full validation of an ITP-ITP-MS method. In order to see the benefits of an online ITP sample pretreatment (preconcentration and clean-up) on the performance parameters, the developed 2D ITP-MS method was compared with a corresponding 1D ITP-MS method. Application potentialities of the compared methods were demonstrated via a determination of two B vitamins, namely thiamine and pyridoxine, in human urine samples. The developed 2D ITP-MS method showed its enhanced effectivity and usefulness for a routine biomedical use (here, a reliable screening of trace B vitamins in human urine without an offline sample preparation).

Ultrasensitive determination of serotonin in human urine by a two dimensional capillary isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry.

A two-dimensional capillary isotachophoresis-capillary zone electrophoresis method hyphenated with tandem mass spectrometry was developed and validated for ultrasensitive quantification of serotonin in real human urine samples. Under optimal conditions, the separation was achieved within 12 min (including on-line sample preparation) with the limit of detection of 34 pg mL-1 (due to a large volume sample injection, here 10 µL, and isotachophoretic preconcentration). This concentration limit represents the lowest value for serotonin in comparison to other previously published separation methods employing mass spectrometry detection and applied to urine matrices. Thanks to high orthogonality, on-line concentration and clean-up effects of this approach, other excellent validation parameters such as linearity (coefficient of determination > 0.99), inter-day and intra-day precision (relative standard deviations 3.5-12.2%), accuracy (relative errors within 99-109.4%), and recovery (96-102%) could be easily obtained too. To demonstrate applicability of the method, we monitored serotonin levels in various real samples (from a healthy volunteer and clinical ones). The determined levels, normalized on the creatinine concentrations, were in the range of 6.81-12.86 ng mmol-1 creatinine This advanced method is suggested for an effective, reliable, high sample throughput, and low cost routine clinical screening or targeted metabolomic studies of serotonin in urine samples.

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study.

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP-CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC-MS/MS, CITP-CITP-CD, and CZE-UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22-2.97% for the creatinine position in analytical profiles), and recovery (87.1-115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing-Bablok regression and Bland-Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC-MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.

Capillary isotachophoresis with electrospray-ionization mass-spectrometric detection: Cationic electrolyte systems in the medium-alkaline range for selective analysis of medium strong bases.

This work extends the present working range of isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection and describes for the first time a functional cationic electrolyte system for analyses at medium-alkaline pH. So far no ITP-MS application was published on the analysis of medium strong bases although there is a broad spectrum of potential analytes like biogenic amines, alkaloids or drugs, where this technique promises interesting gains in both sensitivity and specificity. The presented results include a selection of suitable sufficiently volatile ESI-compatible system components, discussion of factors affecting system properties, and recommendations for functional ITP electrolyte systems. Theoretical conclusions based on calculations and computer simulations are confirmed by experiments with a model mixture of beta-blockers. Practical applicability of the method is demonstrated on the example of analysis of sotalol in dried blood spots where direct injection of aqueous extract, ITP stacking and MS detection provide a fast, simple and sensitive technique with limits of quantitation on the sub-nM level.

Antibody-free detection of amyloid beta peptides biomarkers in cerebrospinal fluid using capillary isotachophoresis coupled with mass spectrometry.

This study reports a capillary isotachophoresis (ITP) - electrospray ionization mass spectrometry (ESI-MS) method for the determination of several amyloid ß (Aß) peptides, which are biomarkers of Alzheimer's disease (AD) in cerebrospinal fluids (CSF). For the first time, these peptides have been detected directly from CSF by MS without recourse to an immunocapture-based sample pre-treatment. The antibody-free approach is based on the marriage between capillary ITP, a powerful on-line electrokinetic preconcentration technique, and MS for simultaneous detection of different Aß peptides. To ensure a good performance, the ITP process of fluorescently labelled Aß peptides was for the first time implemented and verified with laser induced fluorescent detection, prior to methodology transfer to MS detection. Better detection sensitivity was achieved with labelled Aß peptides for both detection modes. Using hydroxyl ions as the terminating and acetate as the leading ions, our method allows efficient ITP preconcentration under alkaline conditions of the slowly migrating Aß peptides to reach quantifiable concentration down to 50 pM. The developed ITP-MS approach allows reliable quantification of different fluorescently derivatized Aß peptides, i.e. Aß 1-42, Aß 1-40 and Aß 1-38 down to sub nM ranges in CSF samples from AD and non-demented (healthy control) patients. Good agreement (<20% deviation) for the determination of Aß 1-42/Aß 1-40 ratio in CSF was achieved between results obtained with this new ITP-MS and our recently developed method based on large volume sample stacking coupled to CE. Discrimination of one AD patient from two healthy controls was successfully made with the Aß 1-42/Aß 1-40 ratio obtained by the developed ITP-MS method for the tested cerebrospinal fluid samples.

Methodology of analysis of very weak acids by isotachophoresis with electrospray-ionization mass-spectrometric detection: Anionic electrolyte systems for the medium-alkaline pH range.

This work describes for the first time a functional electrolyte system setup for anionic isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection in the neutral to medium-alkaline pH range. So far no application was published on the analysis of very weak acids by anionic ITP-MS although there is a broad spectrum of potential analytes with pKa values in the range 5-10, where application of this technique promises interesting gains in both sensitivity and specificity. The problem so far was the lack of anionic ESI-compatible ITP systems in the mentioned pH range as all typical volatile anionic system components are fully ionized at neutral and alkaline pH and thus too fast to suit as terminators. We propose an original solution of the problem based on the combination of two ITP methods: (i) use of the hydroxyl ion as a natural and ESI-compatible terminator, and (ii) use of configurations based on moving-boundary ITP. The former method ensures effective stacking of analytes by an alkaline terminator of sufficiently low mobility and the latter offers increased flexibility for tuning of the separation window and selectivity according to actual needs. A theoretical description of the proposed model is presented and applied to the design of very simple functional electrolyte configurations. The properties of example systems are demonstrated by both computer simulation and experiments with a group of model analytes. Potential effects of carbon dioxide present in the solutions are demonstrated for particular systems. Experimental results confirm that the proposed methodology is well capable of performing sensitive and selective ITP-MS analyses of very weak acidic analytes (e.g. sulfonamides or chlorophenols).

Determination of effective charges and ionic mobilities of polycationic antimicrobial peptides by capillary isotachophoresis and capillary zone electrophoresis.

Capillary ITP (CITP) and CZE were applied to the determination of effective charges and ionic mobilities of polycationic antimicrobial peptides (AMPs). Twelve AMPs (deca- to hexadecapeptides) containing three to seven basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were investigated. Effective charges of the AMPs were determined from the lengths of their ITP zones, ionic mobilities, and molar concentrations, and from the same parameters of the reference compounds. Lengths of the ITP zones of AMPs and reference compounds were obtained from their CITP analyses in cationic mode using leading electrolyte (LE) composed of 10 mM NH4 OH, 40 mM AcOH (acetic acid), pH 4.1, and terminating electrolyte (TE) containing 40 mM AcOH, pH 3.2. Ionic mobilities of AMPs and singly charged reference compounds (ammediol or arginine) were determined by their CZE analyses in the BGE of the same composition as the LE. The effective charges numbers of AMPs were found to be in the range 1.65-5.04, i.e. significantly reduced as compared to the theoretical charge numbers (2.86-6.99) calculated from the acidity constants of the analyzed AMPs. This reduction of effective charge due to tightly bound acetate counterions (counterion condensation) was in the range 17-47% depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5-38.6) × 10-9  m2 V-1 s-1 and in most cases were in a good agreement with the ratio of their effective charges and relative molecular masses.

Comparison of hydrodynamically closed two-dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline.

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un-pretreated urine matrices. An alternative approach, two-dimensional (column-coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low-cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on-line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5-fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.

A novel method for serum lipoprotein profiling using high performance capillary isotachophoresis.

A new capillary isotachophoresis (cITP) method for lipoprotein profiling with superior lipoprotein coverage compared to previous methods has been developed, resolving twice as many lipoprotein species (18 peaks/fractions) in serum or plasma in less than 9.5 min. For this, a novel mixture of 24 spacers, including amino acids, dipeptides and sulfonic acids, was developed and fine-tuned, using predictive software (PeakMaster) and testing of spiked serum samples. Lipoprotein peaks were identified by serum-spiking with reference lipoproteins. Compatibility with common lipophilic stains for selective lipoprotein detection with either UV/Vis or laser-induced fluorescence was demonstrated. A special new capillary with a neutral coating (combining water-compatible OV1701-OH deactivation and methylation) was used for the first time for electrodriven separations, allowing very stable separations in a pH 8.8-9.4 gradient system, being functional for more than 100 injections. Excellent reproducibility was achieved, with coefficients of variation lower than 2.6% for absolute migration times. Comparison was performed with human plasma samples analyzed by NMR, leading to similar results with cITP after multivariate statistics, regarding group-clustering and lipoprotein species correlation. The new cITP method was applied to the analysis of serum samples from a LDL receptor knock-out mice model fed either a normal diet or a western-type diet. Differences in the lipoprotein levels and in the sublipoprotein types were detected, showing a shift to more atherogenic particles due to the high cholesterol diet. In summary, this novel method will allow more detailed and informative profiling of lipoprotein particle subtypes for cardiovascular disease research.

DNA fragment separations by on-line combination of capillary isotachophoresis-capillary zone electrophoresis with UV detection.

A high-speed DNA fragment separation system based on an on-line combination of capillary ITP with CZE (CITP-CZE) and using UV detection at 260 nm was developed. A novel CITP-CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized α-, ß- and γ-cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP-CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1-5 ng/µL (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1-3 and 3-9%, respectively. The developed CITP-CZE system was further applied to the analysis of digest plasmid DNA samples.

Application of isotachophoresis in commercial capillary electrophoresis instrument using C(4) D and UV detection.

In this work, we tested the applicability of a commercial CE instrument (Agilent) for capillary ITP (CITP). The fused silica capillaries were flushed with PVP solution before each sample injection to suppress the EOF. As a dual-detection mode, commercial capacitively coupled contactless conductivity detection and ultraviolet detectors were applied. The experiments showed that the detection gap of the capacitively coupled contactless conductivity detection limits the achievable LOD and the separation resolution when the analyte CITP zones are very narrow, therefore long (120 cm) CE capillary was used and it was largely filled with the sample solution. CITP analyses of several real samples (leather extract, red wine, juice, and fizzy drink) have been demonstrated. In peak mode of CITP when the zone of a chromophore analyte is positioned between nonchromophore zones, excellent sensitivity (in submicromolar concentration range) could be achieved by ultraviolet detection. The hazardous chromate in low concentration was determined in the aqueous extract of tanned leather.

Studies of levels of biogenic amines in meat samples in relation to the content of additives.

The impact of meat additives on the concentration of biogenic amines and the quality of meat was studied. Fresh white and red meat samples were fortified with the following food additives: citric and lactic acids, disodium diphosphate, sodium nitrite, sodium metabisulphite, potassium sorbate, sodium chloride, ascorbic acid, α-tocopherol, propyl 3,4,5-trihydroxybenzoate (propyl gallate) and butylated hydroxyanisole. The content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine and 2-phenylethylamine was determined by capillary isotachophoretic methods in meat samples (fresh and fortified) during four days of storage at 4°C. The results were applied to estimate the impact of the tested additives on the formation of biogenic amines in white and red meat. For all tested meats, sodium nitrite, sodium chloride and disodium diphosphate showed the best inhibition. However, cadaverine and putrescine were characterised by the biggest changes in concentration during the storage time of all the additives. Based on the presented data for the content of biogenic amines in meat samples analysed as a function of storage time and additives, we suggest that cadaverine and putrescine have a significant impact on meat quality.

Enantioselective column coupled electrophoresis employing large bore capillaries hyphenated with tandem mass spectrometry for ultra-trace determination of chiral compounds in complex real samples.

A new multidimensional analytical approach for the ultra-trace determination of target chiral compounds in unpretreated complex real samples was developed in this work. The proposed analytical system provided high orthogonality due to on-line combination of three different methods (separation mechanisms), i.e. (1) isotachophoresis (ITP), (2) chiral capillary zone electrophoresis (chiral CZE), and (3) triple quadrupole mass spectrometry (QqQ MS). The ITP step, performed in a large bore capillary (800 µm), was utilized for the effective sample pretreatment (preconcentration and matrix clean-up) in a large injection volume (1-10 µL) enabling to obtain as low as ca. 80 pg/mL limits of detection for the target enantiomers in urine matrices. In the chiral CZE step, the different chiral selectors (neutral, ionizable, and permanently charged cyclodextrins) and buffer systems were tested in terms of enantioselectivity and influence on the MS detection response. The performance parameters of the optimized ITP - chiral CZE-QqQ MS method were evaluated according to the FDA guidance for bioanalytical method validation. Successful validation and application (enantioselective monitoring of renally eliminated pheniramine and its metabolite in human urine) highlighted great potential of this chiral approach in advanced enantioselective biomedical applications.

Comparison of hydrodynamically closed isotachophoresis-capillary zone electrophoresis with hydrodynamically open capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: pheniramine, its metabolite and phenylephrine in human urine.

The advanced two dimensional isotachophoresis (ITP)-capillary zone electrophoresis (CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed in this work to demonstrate analytical potentialities of this approach in the analysis of drugs in multicomponent ionic matrices. Pheniramine (PHM), phenylephrine (PHE), paracetamol (PCM) and their potential metabolic products were taken for the analysis by the ITP-CZE-ESI-QqQ technique working in hydrodynamically closed CE separation system and then a comparison with the conventional (hydrodynamically open) CZE-ESI-QqQ technique was made. The ITP-CZE-ESI-QqQ method was favorable in terms of obtainable selectivity (due to highly effective heart-cut analysis), concentration limits of detection (LOD at pgmL(-1) levels due to enhanced sample load capacity and ITP preconcentration), sample handling (on-line sample pretreatment, i.e. clean-up, preconcentration, preseparation), and, by that, possibilities for future automation and miniaturization. On the other hand, this experimental arrangement, in contrast to the CZE-ESI-QqQ arrangement supported by an electroosmotic flow, is principally limited to the analysis of uniformly (i.e. positively or negatively) charged analytes in one run without any possibilities to analyze neutral compounds (here, PCM and neutral or acidic metabolites of the drugs had to be excluded from the analysis). Hence, these general characteristics should be considered when choosing a proper analytical CE-MS approach for a given biomedical application. Here, the analytical potential of the ITP-CZE-ESI-QqQ method was demonstrated showing the real time profiles of excreted targeted drugs and metabolite (PHM, PHE, M-PHM) in human urine after the administration of one dose of Theraflu(®) to the volunteers.

On-line column coupled isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: varenicline and its metabolite in human urine.

A new highly advanced analytical approach, based on two-dimensional column coupled CE (ITP-CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed, evaluated and applied in biomedical field in the present work. Capillary isotachophoresis (ITP) coupled on-line with capillary zone electrophoresis (CZE) used in hydrodynamically closed separation system was favorable for increasing the sample load capacity, increasing the analyte concentration, and removing the deteriorative highly conductive major matrix constituents. These factors considerably reduced the concentration limits of detection (cLOD) and external sample preparation (comparing to single column CZE), and, by that, provided favorable conditions for the mass spectrometry (enhanced signal to noise ratio, reproducibility of measurements, working life of MS). Here, the CZE-ESI combination provided more effective interfacing than ITP-ESI resulting in both a higher obtainable intensity of MS detection signal of the analyte as well as reproducibility of measurements of the analyte's peak area. The optimized ITP-CZE-ESI-QqQ method was successfully evaluated as for its performance parameters (LOD, LOQ, linearity, precision, recovery/accuracy) and applied for the direct identification and ultratrace (pgmL(-1)) determination of varenicline and, in addition, identification of its targeted metabolite, 2-hydroxy-varenicline, in unpretreated/diluted human urine. This application example demonstrated the real analytical potential of this new analytical approach and, at the same time, served as currently the most effective routine clinical method for varenicline.

Analytical approaches to determination of carnitine in biological materials, foods and dietary supplements.

l-Carnitine is a vitamin-like amino acid derivative, which is an essential factor in fatty acid metabolism as acyltransferase cofactor and in energy production processes, such as interconversion in the mechanisms of regulation of cetogenesis and termogenesis, and it is also used in the therapy of primary and secondary deficiency, and in other diseases. The determination of carnitine and acyl-carnitines can provide important information about inherited or acquired metabolic disorders, and for monitoring the biochemical effect of carnitine therapy. The endogenous carnitine pool in humans is maintained by biosynthesis and absorption of carnitine from the diet. Carnitine has one asymmetric carbon giving two stereoisomers d and l, but only the l form has a biological positive effect, thus chiral recognition of l-carnitine enantiomers is extremely important in biological, chemical and pharmaceutical sciences. In order to get more insight into carnitine metabolism and synthesis, a sensitive analysis for the determination of the concentration of free carnitine, carnitine esters and the carnitine precursors is required. Carnitine has been investigated in many biochemical, pharmacokinetic, metabolic and toxicokinetic studies and thus many analytical methods have been developed and published for the determination of carnitine in foods, dietary supplements, pharmaceutical formulations, biological tissues and body fluid. The analytical procedures presented in this review have been validated in terms of basic parameters (linearity, limit of detection, limit of quantitation, sensitivity, accuracy, and precision). This article presented the impact of different analytical techniques, and provides an overview of applications that address a diverse array of pharmaceutical and biological questions and samples.

Quantitative capillary zone electrophoresis method for the precise determination of charge differences arising from the manufacture of heparan-N-sulfatase.

A rapid and reproducible high-resolution capillary zone electrophoresis (CZE) method capable of resolving the charge isoforms of intact heparan-N-sulfatase (HNS) has been developed to monitor the charge consistency across different batches of HNS. Separation was carried out using a bare fused silica capillary with a buffer system composed of 25 mM Tris, pH 8.0. This CZE method allowed the separation and integration of 14 peaks, each arising from differences in the amount of sialic-acid and mannose-6-phosphate bearing glycoforms, which were confirmed using enzymatically modified samples. Standard conditioning and rinsing conditions of the capillary were used to achieve optimal repeatability. Excellent day-to-day precision was obtained for migration times of each peak relative to the electroosmotic flow marker with relative standard deviation (RSD)≤ 0.5%. The precision of the relative peak areas (peak area percentages) ranged from 0.6% to 2.8% RSD for the major isoforms (peaks 3-12), from 4.0% to 5.0% RSD for peaks 1 and 2, and from 7.4% to 23.2% RSD for peaks 13 and 14. The method was able to discriminate charge variation across different batches of HNS, including those with both significant and minor process changes.