A fast, single column high-performance anion exchange chromatography with pulsed amperometric detection method for the determination of saccharides in atmospheric aerosol samples.

Saccharides, especially anhydro sugars present in atmospheric aerosols, can be used as tracers to track sources of atmospheric aerosols. High-performance anion-exchange chromatography with pulsed amperometric detection is a commonly used technique for determining these saccharides, but the reported methods suffer from three drawbacks. First, to achieve separation of the complete set of atmospheric saccharides, run times are very long, typically longer than 60 minutes. Second, some methods require two columns to achieve the desired separation. Finally, in an era when electrolytic eluent preparation allows for excellent precision and accuracy, these methods require manually prepared eluents, which can lead to separation inconsistency for closely eluting analytes. These drawbacks make existing methods difficult to automate. To address this issue, we developed a fast method that uses only a single column for separation and electrolytically generated eluent that resolves 12 key atmospheric aerosol saccharides in 20 minutes. The resolved saccharides include anhydro sugars (levoglucosan, galactosan, and mannosan), sugar alcohols (erythritol, xylitol, and mannitol), and mono-/disaccharides (arabinose, galactose, glucose, mannose, fructose, and sucrose). To our knowledge, this report is the first instance of achieving such a significant reduction in run time with good resolution for this set of saccharides.

An improved method for galactosyl oligosaccharide characterization.

Due to beneficial effects of galactosyl oligosaccharides (GOS) on digestive and immune health, their characterization has become increasingly important. This is especially so as GOS are synthesized enzymatically and contain oligosaccharides of different sizes and linkages. High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is widely used for GOS characterization. With its high resolving power, it can separate structural isomers. Here we present a significant improvement to currently used methods. Our approach combines high resolution HPAE separation on a CarboPac PA300 column with 4 µm particle size with PAD and Orbitrap mass spectrometry (MS) detections to provide in-depth information on GOS composition. Oligosaccharide resolution, especially in the disaccharide region, is significantly improved and can be routinely achieved. Improvement in technology to remove sodium before MS results in minimal peak dispersion, allowing GOS degrees of polymerization 2 to 6 to be identified based on mass spectra obtained from intact oligosaccharides and confirmed using fragmentation patterns observed in MS/MS data. Combining HPAE with MS led to identification of 28 oligosaccharides in a commercial GOS sample. We attempted to correlate oligosaccharide structure with observed elution behavior. To our knowledge this is first such attempt and can form a basis for a comprehensive structure vs HPAE elution behavior database.

Determination of dimethylamine and nitrite in pharmaceuticals by ion chromatography to assess the likelihood of nitrosamine formation.

Since July 2018 several drugs have been recalled due to contamination with N-nitrosodimethylamine (NDMA), a probable human carcinogen. Dimethylamine (DMA) and nitrite are precursors in the formation of NDMA. In this study, ion chromatography (IC) methods were developed for the determination of these two precursors in drug substances and drug products. Two methods were developed to determine DMA in two drug products using a cation exchange separation coupled to suppressed conductivity detection. The limit of detection of DMA is < 1 µg/g of active pharmaceutical ingredient (API) for both methods. Nitrite was determined using an anion exchange separation coupled with UV absorbance detection. The limit of detection of nitrite was 0.918 µg/g API. The developed methods were successfully applied to DMA and nitrite determinations in five drug products including metformin, losartan, ranitidine, Nytol, and Benadyrl, and two drug substances (APIs), losartan potassium and metformin hydrochloride. Some samples contained nitrite and DMA at detectable levels. Dimethylamine and nitrite recovery from pharmaceutical samples ranged from 96.0-104 %. The developed methods should be useful for the rapid screening and quantification of nitrite and DMA in pharmaceuticals and in-process samples to assess the likelihood of NDMA formation. The methods for DMA should be applicable to other amines to assess the likelihood of the formation of other nitrosamines in pharmaceutical products.

Targeted Quantitation Mode Comparison of Haloacetic Acids, Bromate, and Dalapon in Drinking Water Using Ion Chromatography Coupled to High-Resolution (Orbitrap) Mass Spectrometry.

A highly selective, sensitive, and simple analytical method for identification and quantification of nine haloacetic acids, bromate, and dalapon has been developed. This method uses ion chromatography (IC) coupled with electrospray ionization-high-resolution mass spectrometry (IC-ESI-HRMS) to directly analyze water samples on a high capacity anion-exchange column, eliminating the need for sample pretreatment/derivatization. Our study compared the following three types of targeted quantitation experiments using a quadrupole-orbitrap hybrid mass spectrometer, full-scan MS with data-dependent tandem mass spectrometry (full MS/dd-MS2 with inclusion list), targeted selected ion monitoring (SIM) with data-dependent tandem mass spectrometry (t-SIM/dd-MS2), and parallel reaction monitoring (PRM). Sensitivity, linearity, accuracy, and precision were validated following the guidelines of U.S. EPA Method 557. Single laboratory lowest concentration minimum reporting levels (LCMRLs) for the analytes using three different acquisition modes ranged from 0.0011 to 0.18 µg/L. All three quantitation modes showed good linearity for the eleven analytes with coefficients of determination of 0.9981- 0.9993. This IC-ESI-HRMS method was successfully applied to the analysis of commercial bottled water, tap water from San Francisco Bay Area, and the same tap water that has been through a filtered drinking water faucet. Both t-SIM/dd-MS2 and PRM modes were sensitive to confirm the trace-level presence of all nine HAAs, bromate, and dalapon in the tap water sample. Full-scan HRMS data acquisition provided the benefits of simultaneous data collection for both targeted and non-targeted components, and thus, suitability for simultaneous quantification of an unlimited number of compounds. Data-dependent MS/MS (dd-MS2) product-ion spectra were used for confirmation. All three modes showed good quantitative performance and obtained similar values. Single laboratory precision and accuracy data are presented for three water matrices: reagent water, laboratory synthetic sample matrix (LSSM), and tap water. Single laboratory precision was 0.078- 8.04%, and accuracy was in the range 70-130% for the three MS modes.

Selective and sensitive determination of bromate in bread by ion chromatography-mass spectrometry.

Potassium bromate is a food additive used as "flour improver" in the baking industry. Bromate is considered a carcinogenic and nephrotoxic substance. Thus, the bromate concentration must be carefully monitored in flour products. We developed a method for a selective and sensitive determination of bromate in flour that uses ion chromatography coupled with single quadrupole mass spectrometry (IC -MS). A recently introduced high-capacity anion-exchange column was used to separate bromate from matrix anions. Six commercial flour and flour products including homemade bread baked using flour containing potassium bromate, were analyzed. The method showed good precision with RSDs <0.2%, and <5% (n = 8), for retention time and peak area respectively. Bromate recoveries from flour samples ranged from 86 to 110%. The limits of detection and quantitation of bromate in the prepared solution were 0.10 µg/L and 0.34 µg/L, respectively, which corresponded to 5 µg/kg and 17 µg/kg in bread.

Vaccine Quality Ensured by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection.

Many important vaccines use bacterial capsular polysaccharides, or shorter polysaccharides or oligosaccharides, derived from the capsular polysaccharides, conjugated to protein. It is imperative that manufacturers understand the carbohydrate composition of these vaccines and deliver a product with a consistent polysaccharide or polysaccharide conjugate composition and content. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is a major technique used to understand the carbohydrate composition of these vaccines and ensure product quality. HPAE-PAD separates and detects carbohydrates without analyte derivatization. This paper describes the basics of the HPAE-PAD technique and then reviews how it has been applied to Haemophilus influenzae type b, pneumococcal, meningococcal, group B streptococcal, and Salmonella polysaccharide and corresponding conjugate vaccines.

Profiling N-linked oligosaccharides from IgG by high-performance anion-exchange chromatography with pulsed amperometric detection.

Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For example, high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation. The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations (RSDs) were between 0.63 and 4.0% while retention indices precision RSDs ranged from 0.27 to 0.56%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.

Simultaneous determination of total nitrogen and total phosphorus in environmental waters using alkaline persulfate digestion and ion chromatography.

An ion chromatography (IC) method was developed for the simultaneous determination of total nitrogen and total phosphorus after alkaline persulfate digestion. This study takes advantage of advances in construction of high-resolution, high-capacity anion-exchange columns that can better tolerate the matrices typically encountered when a determination of total nitrogen and total phosphorous is required. Here, we used an electrolytically generated hydroxide eluent combined with a high-capacity, hydroxide-selective, anion-exchange column for the determination of total nitrogen (as nitrate-N) and total phosphorus (as phosphate-P) in environmental samples by IC. This method yielded LODs for nitrate-N and phosphate-P of 1.0 and 1.3 µg/L, respectively. The LOQs determined for these analytes were 3.4 and 4.2 µg/L, respectively. Due to the dilution factor required and the blank nitrate-N concentration after the persulfate digestion, the quantification limits increased for nitrate-N and phosphate-P to 171 and 63 µg/L, respectively. The suitability of the method was evaluated by determining the nitrogen and phosphorus concentrations from known concentrations of organic-containing nitrogen and phosphorus compounds. In addition, environmental samples consisting of six different wastewaters and 48 reservoir samples were evaluated for total nitrogen and phosphorus. The recoveries of nitrogen and phosphorus from the organic-containing compounds ranged from 93.1 to 100.1% and 85.2 to 97.1%, respectively. In addition, good correlation between results obtained by the colorimetric method and IC was also observed. The linearity, accuracy, and evaluation of potential interferences for determining TN and TP will be discussed.

Evaluation of desialylation during 2-amino benzamide labeling of asparagine-linked oligosaccharides.

Labeling of released asparagine-linked (N-linked) oligosaccharides from glycoproteins is commonly performed to aid in the separation and detection of the oligosaccharide. Of the many available oligosaccharide labels, 2-amino benzamide (2-AB) is a popular choice for providing a fluorescent product. The derivatization conditions can potentially lead to oligosaccharide desialylation. This work evaluated the extent of sialic acid loss during 2-AB labeling of N-linked oligosaccharides released from bovine fetuin, polyclonal human serum immunoglobulin G (IgG), and human α1-acid glycoprotein (AGP) as well as of sialylated oligosaccharide reference standards and found that for more highly sialylated oligosaccharides the loss is greater than the <2% value commonly cited. Manufacturers of glycoprotein biotherapeutics need to produce products with a consistent state of sialylation and, therefore, require an accurate assessment of glycoprotein sialylation.

Modernizing the assay for ammonia in pharmaceuticals.

The United States Pharmacopeia USP35-NF30 contains monographs for adenosine and sodium bicarbonate with imprecise and outdated color comparison assays for ammonia. In the spirit of monograph modernization we developed ion chromatography (IC) assays for ammonia that meet the requirements of the adenosine and sodium bicarbonate monographs. Ammonia, as ammonium, in a solution of adenosine is separated on a high-performance cation-exchange column and detected by suppressed conductivity. This assay requires <5min per sample and accurately determines the ammonia content of adenosine. For sodium bicarbonate a different IC method was required to determine ammonia due to the high concentration of sodium relative to ammonia in a sodium bicarbonate solution. This assay uses a high-capacity cation-exchange column that has the appropriate selectivity for determining low concentrations of ammonia in the presence of high concentrations of sodium. For both the methods, the precisions (retention time RSD<0.1%, peak area RSD<2.3%), accuracy (average recovery 70-117), limits of detection (LOD) and quantitation (LOQ), and robustness were measured according to the analytical performance requirements described in USP General Chapter <1225>.

Interlaboratory study on differential analysis of protein glycosylation by mass spectrometry: the ABRF glycoprotein research multi-institutional study 2012.

One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods.

Choline in infant formula and adult nutritionals by ion chromatography and suppressed conductivity: First Action 2012.20.

Single-laboratory validation (SLV) data from a method for the determination of choline in infant formula and adult nutritionals by ion chromatography (IC) and suppressed conductivity were generated and presented to the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) at the AOAC Annual Meeting held in Las Vegas, NV, during September 30 to October 3, 2012. The ERP reviewed the data and concluded that the data met the standard method performance requirements (SMPRs) established and approved the method as AOAC Official First Action. At the ERP's request, a second, full SLV was performed on 17 SPIFAN matrixes that included fortified and placebo products. Prior to IC analysis, microwave-assisted acid hydrolysis was used to digest and release bound choline from powder and ready-to-feed (RTF) infant formula and adult nutritional samples. Following hydrolysis, separation of choline from common cations was achieved on a Thermo Scientific Dionex IonPac CS19 column followed by suppressed conductivity detection. Total choline was measured and reported as the choline ion in mg/100 g reconstituted material or RTF as-is. The system was calibrated over the analytical range specified in the SMPR (2-250 mg/100 g). Recoveries of spiked samples at 50 and 100% of the fortified choline amounts ranged from 93.1 to 100.7% with RSDs < or = 6.7% for product containing < 2 mg/100 g and < or = 4.1% for product containing 2-100 mg/100 g. Accuracy for the National Institute of Standards and Technology Standard Reference Material 1849a was determined over a 6-day interval and found to be 10.2 +/- 0.2 mg/100 g calculated as the reconstituted powder with an RSD of 1.8%. The LOD was determined to be 0.009, and the LOQ 0.012 mg/100 g, well below the SMPR requirements of 0.7 and 2 mg/100 g, respectively. Repeatability RSDs over the range of the assay (2-200 mg/100 g) ranged from 1.0 to 5.93%

Five-minute glycoprotein sialic acid determination by high-performance anion exchange chromatography with pulsed amperometric detection.

Glycoprotein sialylation analysis is a common analytical step in characterizing biotherapeutic products and expression experiments to optimize production. In this article, a high-throughput (5-min) high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD)-based analytical method for glycoprotein sialic acid determination is described. Results from this method are compared with both published HPAE-PAD and 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatization followed by ultra high-performance liquid chromatography fluorescence detection (UHPLC-FLD) assays. The quantified sialic acid amounts agree with prior HPAE-PAD analyses within replicate error and with UHPLC-FLD within an average of 24%, which are equivalent results based on assay reproducibility.

Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

Development and validation of an assay for iodide in serum using ion chromatography with pulsed amperometric detection.

We developed and validated an ion chromatography method to assay iodide in serum sampled from rats and rabbits that had been exposed to iodomethane. Iodomethane is of interest because it is a volatile liquid pre-plant soil crop protection fumigant that has been proposed as a non-ozone-depleting alternative to methyl bromide. Serum was prepared from whole blood collected on wet ice at the time of sacrifice and kept frozen at less than -65 degrees C. For analysis, serum samples were thawed unassisted at ambient temperature. Proteins were separated from the serum samples by ultrafiltration. A 100-microl filtered serum sample was then injected into the ion chromatograph without additional sample preparation. Iodide was separated in <20 min by anion-exchange chromatography using a 25-mM nitric acid eluent. The analyte of interest was detected by pulsed amperometry using a silver working electrode. The method showed linear response over the concentration range of 100 to 5000 ng/ml iodide (r2>.998) with a lower limit of quantitation of 100 ng/ml iodide. The accuracy of the procedure, determined by spiked recovery measurements at 100 ng/ml iodide, was between 90 and 110%. A method detection limit of 20 ng/ml for iodide in serum samples was demonstrated using the method of standard additions.

Identification of tobramycin impurities for quality control process monitoring using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

Commercial-scale fermentation for tobramycin manufacture is carried out with Streptomyces tenebrarius. Impurity profiling during various phases of pharmaceutical production is important for evaluating the effectiveness of a processing step and meeting regulatory requirements. High-performance anion-exchange (HPAE) chromatography with integrated pulsed amperometric detection (HPAE-IPAD) is a highly sensitive method used to assay tobramycin and to assess purity, but no prior publications demonstrated the capability of this technique to monitor purity at various stages of production at either the typical concentrations or in the typical matrices of a manufacturing process. In addition, the identities of the impurity peaks observed in commercial sources of tobramycin when assayed by using HPAE-IPAD are mainly unknown. Regulatory agencies generally require these impurities to be characterized when found above certain limits, and when present at higher levels require toxicological studies. In this paper, we analyze tobramycin samples using HPAE-IPAD at different stages of production and show the impurity profile and concentration changes through the manufacturing process. We successfully identified nearly all the impurity peaks found in commercially available tobramycin, based on known degradation pathways deduced from extreme pH forced degradation studies, which we experimentally reproduced, and based on previously known related substances found in S. tenebrarius fermentation broth. In crude and final tobramycin products, we identified the peaks for neamine, kanamycin B, nebramine, kanosamine, 2-deoxystreptamine. We tentatively identified deoxystreptamine-kanosaminide in crude and final products, and kanamycin A, carbamoyl-kanamycin B and carbamoyl-tobramycin in down stream process intermediates of a S. tenebrarius fermentation culture. Results presented in this paper support the effective use of the HPAE-IPAD method for in-process impurity profiling of tobramycin, and as a stability-indicating technique after product purification.

Direct determination of free cyanide in drinking water by ion chromatography with pulsed amperometric detection.

Cyanide is a regulated contaminant in drinking water in the United States. This paper describes an ion chromatography method with pulsed amperometric detection (PAD) that directly determines free cyanide in drinking water. Samples are treated with sodium hydroxide to stabilize cyanide and with a cation-exchange cartridge to remove transition metals. Cyanide is separated by anion-exchange chromatography and detected by PAD with a waveform optimized for cyanide and used with a disposable silver working electrode. The recovery of cyanide spiked into five water samples was >80%. With an MDL of 1.0 microg/L, this method determines cyanide concentrations well below the reporting limits for free cyanide in drinking water.

Determination of neomycin sulfate and impurities using high-performance anion-exchange chromatography with integrated pulsed amperometric detection.

Neomycin B is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that neomycin sulfate and its major impurities, including neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.40 mM) at a column temperature of 30 degrees C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between neomycin B and the closest major impurity ranged from 6.56 and 7.45 over 10 days of consecutive analysis (7.24+/-0.10, n=836 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (10 days, 822 injections) retention time stability with a R.S.D. of 0.6%. Peak area R.S.D. (10 microM) was 1.3%. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. The spike recoveries of neomycin B from extractions of three different topical ointments and cream formulations ranged from 95 to 100%. The measured concentration of neomycin B in these formulations ranged from 119 to 154% of the label concentration. The R.S.D. for the measured concentration of one of the formulations tested over three separate days, n=11 extracts, was 3.2%. Based on the results of these evaluations, we believe this method can be used for neomycin sulfate identity, assay, and purity.

Determination of tobramycin and impurities using high-performance anion exchange chromatography with integrated pulsed amperometric detection.

Tobramycin is one of a class of aminoglycoside antibiotics that lack a good chromophore, and is therefore difficult to determine using reversed-phase HPLC with absorbance detection. This is especially true for determining the quantity of each impurity. We show that tobramycin and its major impurities, including kanamycin B and neamine (neomycin A), can be separated on a strong anion-exchange column using a weak potassium hydroxide eluent (2.00 mM) at a column temperature of 30 degrees C, and directly detected by integrated pulsed amperometric detection (IPAD). The resolution (United States Pharmacopeia (USP) definition) between tobramycin and kanamycin B ranged from 5.71 and 6.06 over 7 days of consecutive analysis (5.92+/-0.07, n = 590 injections). Due to the difficulty of producing weak hydroxide eluents of the required purity (i.e. carbonate-free), this method depends on automatic eluent generation to ensure method ruggedness. This method exhibited good long-term (50 days, 2368 injections) retention time stability with R.S.D.s of 0.4% and 0.3% for tobramycin and kanamycin B, respectively. Peak area R.S.D.s for tobramycin and kanamycin B (10 microM each, 20 microL injection) over 7 days (572 injections) were 2.3% and 1.9%, respectively. Method robustness was evaluated by intentionally varying the flow rate, eluent concentration, column temperature, and column. Based on the results of these evaluations, this method can be used for tobramycin identity, assay, and purity.