Evaluation of Busulfan as a Third-Party Immunoassay on a Clinical Chemistry Analyzer.

BACKGROUND: Busulfan is widely used in conditioning regimens to prepare patients for hematopoietic stem cell transplantation. Therapeutic drug monitoring (TDM) is critical due to large inter- and intra-individual variability in busulfan pharmacokinetics, and the risk of adverse consequences of toxicity including hepatic veno-occlusive disease. Busulfan is most commonly measured by liquid chromatography-mass spectrometry (LC-MS/MS), which is not as widely available in clinical laboratories as automated routine clinical chemistry analyzers. The objective was to perform analytical verification of a busulfan immunoassay on the Abbott Alinity c platform. METHODS: The MyCare Oncology busulfan immunoassay was configured as a third-party reagent on the Abbott Alinity c. Imprecision, linearity, sample carryover, and onboard stability of reagent studies were evaluated. The performance of the busulfan immunoassay using the Abbott Alinity c was compared to the Beckman Coulter AU480 using sodium heparinized plasma, as well as to LC-MS/MS using lithium heparinized plasma. RESULTS: The imprecision goal of 8% was met, and linearity within the analytical measurement range of 240 to 1700 ng/mL was verified. Sample carryover was negligible, and the reagents were stable onboard for at least 84 days. The busulfan immunoassay correlated well with LC-MS/MS (slope = 0.949, y-intercept = -7.8 ng/mL, r2 = 0.9935) and the Beckman Coulter AU480 (slope = 1.090, y-intercept = -34.5 ng/mL, r2 = 0.9988). CONCLUSIONS: This study demonstrated successful analytical verification of a busulfan third-party immunoassay on the Abbott Alinity c platform. The ability to perform TDM of busulfan on a routine clinical chemistry analyzer will positively impact turnaround times to improve patient outcomes.

Discovery and early development of non-suppressed ion chromatography.

This year marks the 30th anniversary of the publication of Non-Suppressed Ion Chromatography, which is a method for the rapid separation of anions with on-line conductimetric detection. In this method, the separation column is connected directly to the conductimetric detector. This single-column method is a simpler technique than the original suppressed ion chromatography method, which requires a large suppressor column to reduce the background conductance. In the new method, the background signal is reduced to a manageable level simply by using an ion-exchange separation column of low exchange capacity that lowers the eluent concentration needed for separation. The eluent ion used for separation is chosen based on having large, bulky structure, which lowers the equivalent conductance and facilitates detection of the sample anions. This is a personal account of the initial discovery and early development of non-suppressed ion chromatography. The circumstances for the discovery are recounted by the two authors. Methods are described for determination of anions, cations with indirect detection, and techniques for increasing detection sensitivity. A fundamental equation for the prediction of ion chromatography detector response is given, and the development of several types of detection schemes for ion chromatography is discussed. Finally, the impact of non-suppressed ion chromatography is discussed together with comments on the discovery process.

A rapid, simple method for determining formaldehyde in drinking water using colorimetric-solid phase extraction.

Formaldehyde has been detected in drinking water supplies across the globe and on board NASA spacecraft. A rapid, simple, microgravity-compatible technique for measuring this contaminant in water supplies using colorimetric-solid phase extraction (C-SPE) is described. This method involves collecting a water sample into a syringe by passage through a cartridge that contains sodium hydroxide, to adjust pH, and Purpald, which is a well-established colorimetric reagent for aldehydes. After completing the reaction in the syringe by agitating for 2 min on a shaker at 400 rpm, the 1.0-mL alkaline sample is passed through an extraction disk that retains the purple product. The amount of concentrated product is then measured on-disk using diffuse reflectance spectroscopy, and compared to a calibration plot generated from Kubelka-Munk transformations of the reflectance data at 700 nm to determine the formaldehyde concentration. This method is capable of determining formaldehyde concentrations from 0.08 to 20 ppm with a total work-up time of less than 3 min using only 1-mL samples.

Separation of organic cations using novel background electrolytes by capillary electrophoresis.

A background electrolyte for capillary electrophoresis containing tris(-hydroxymethyl) aminomethane (THAM) and ethanesulfonic acid (ESA) gives excellent efficiency for separation of drug cations with actual theoretical plate numbers as high as 300,000. However, the analyte cations often elute too quickly and consequently offer only a narrow window for separation. The best way to correct this is to induce a reverse electroosmotic flow (EOF) that will spread out the peaks by slowing their migration rates, but this has always been difficult to accomplish in a controlled manner. A new method for producing a variable EOF is described in which a low variable concentration of tributylammonium- or triethylammonium ESA is added to the BGE. The additive equilibrates with the capillary wall to give it a positive charge and thereby produce a controlled opposing EOF. Excellent separations of complex drug mixtures were obtained by this method.

Determination of glycerophosphate and other anions in dentifrices by ion chromatography.

Simple, reliable and sensitive analytical methods to determine the anions, such as fluoride, monofluorophaosphate, glycerophosphate related to anticaries are necessary for basic investigations of anticaries and quality control of dentifrices. A method for the simultaneous determination of organic acids, organic anions and inorganic anions in the sample of commercial toothpaste is proposed. Nine anions (fluoride, chloride, nitrite, nitrate, sulfate, phosphate, monofluorophaosphate, glycerophosphate and oxalic acid) were analyzed by means of ion chromatography using a gradient elution with KOH as mobile phase, IonPac AS18 as the separation column and suppressed conductivity detection. Optimized analytical conditions were further validated in terms of accuracy, precision and total uncertainty and the results showed the reliability of the IC method. The relative standard deviations (RSD) of the retention time and peak area of all species were less than 0.170 and 1.800%, respectively. The correlation coefficients for target analytes ranged from 0.9985 to 0.9996. The detection limit (signal to noise ratio of 3:1) of this method was at low ppb level (<15 ppb). The spiked recoveries for the anions were 96-103%. The method was applied to toothpaste without interferences.

Ultrafast concentration and speciation of chromium(III) and (VI).

There is an increasing need to know the concentrations of chromium(III) and (VI) separately rather than only the total chromium content. A method is described for accomplishing this very quickly using only low-cost, portable equipment. Two small, resin-loaded extraction disks are placed one on top of the other in a plastic holder. Then a syringe containing the aqueous sample is attached to the holder and the sample is pushed through the disks. In a matter of seconds, all of the chromium(VI) is retained on the top anion-exchange disk and chromium(III) is extracted by the second cation-exchange disk. The concentrations on each disk are several hundredfold higher than they were in the original sample. The amounts of chromium(III) and (VI) extracted are measured directly on the surface of the respective disks by diffuse reflectance spectroscopy (DRS). Despite the low molar absorptivity of chromium(III) in aqueous solution, the concentration on the upper most layer on the extraction disk is high enough to permit the determination of chromium(III) in samples at the low mg/L range. Chromium(VI) can also be determined at low to sub-mg/L concentrations. A study of the cation-exchange disks was undertaken to compare the performance characteristics of disks containing sulfonated resins and those with iminodiacetate functionality. In addition, data are presented to show the effects of heating the iminodiacetate disks after the initial extraction. The disks were heated in hot water for 15-30 min to complete the slow complexation reaction on the surface.

Single-column method of ion chromatography for the determination of common cations and some transition metals.

A single-column method for the simultaneous determination of common cations and transition metals in real samples is proposed in this paper. Eleven cations (copper, lithium, sodium, ammonium, potassium, cobalt, nickel, magnesium, calcium, strontium and zinc) were separated and analyzed by means of ion chromatography using an isocratic elution with 2.5 mM methane sulfonic acid and 0.8 mM oxalic acid as mobile phase, IonPac SCS1 (250 mm x 4 mm I.D.) as the separation column and non-suppressed conductor detection. Optimized analytical conditions were further validated in terms of accuracy, precision and total uncertainty and the results showed the reliability of the IC method. The relative standard deviations (RSDs) of the retention time and peak area were less than 0.04 and 1.30%, respectively. The coefficients of determination for cations ranged from 0.9988 to 1.000. The method developed was successfully applied to determination of cations in samples of beer and bottled mineral water. The spiked recoveries for the cations were 94-106%. The method was applied to beer and beverage without interferences.

Metathesis polymerization-derived monolithic membranes for solid-phase extraction coupled with diffuse reflectance spectroscopy.

Novel monolithic disks were prepared via ring opening metathesis polymerization (ROMP) from norborn-2-ene (NBE), a crosslinker, i.e., 1,4,5,8,8a-hexahydro-1,4,4,5,8, exo, endo-dimethanonaphthalene (DMN-H6) and tris(norborn-5-ene-2-ylmethylenoxy)methylsilane, respectively, 2-propanol and toluene (25:25:41:9, all %, w/w) using RuCl2(PCy3)2(CHPh) (Cy=cyclohexyl) as initiator and triphenylphosphine (PPh3) as modulator. Disks 1-2 mm thick were prepared by polymerization in a mold, disks thinner than 1mm were prepared by impregnation of nylon or other porous filters prior to the polymerization step. These disks were evaluated for the preconcentration of iodine and selected organic solutes from dilute aqueous samples by solid-phase extraction (SPE). Quantitative measurement of the extracted solutes was achieved by diffuse-reflectance spectroscopy (DRS) directly on the surface of the disk.

Ion association with alkylammonium cations for separation of anions by capillary electrophoresis.

A background electrolyte (BGE) containing a 100 mM concentration of an alkylammonium cation with ethyl, propyl or butyl groups provides an excellent medium for separation of anions by capillary electrophoresis (CE). Two major effects were noted. Use of one of a series of alkylammonium cations in the BGE at a selected pH provides a simple and effective way to vary and control electroosmotic flow (EOF) over a broad range. It is believed that the alkylammonium cations are coated onto the capillary surface through a reversible dynamic equilibrium. Secondly, alkylammonium cations modify the electrophoretic migration of sample anions and the electroosmotic migration of neutral organic analytes by association interaction. This selective interaction results in improved anion separations and permits the simultaneous separation of neutral analytes. The degree of association interaction varies with the bulk and hydrophobicity of the alkylammonium cations. Incorporation of an aliphatic amine salt of moderate molecular weight in the running electrolyte provides a valuable new way to vary the migration times of sample anions and to optimize their resolution. The interactions between alkylammonium cations and sample anions or neutral organics appear to take place entirely within the liquid phase and do not require a polymeric or micellar pseudo phase.

Factors affecting selectivity in ion chromatography.

Methods for separation of ions by ion-exchange, ion-pair, and zwitterion ion chromatography share at least one common thread--the induced formation of a cation-anion pair in the stationary phase. Selectivity can be defined as the relative ability of sample ions to form such a pair. Examples are given in anion-exchange chromatography to show the effect of variations in the geometry, bulkiness and polarity of the resin cation on selectivity. The type of resin matrix, the hydrophobic nature of the resin surface and the degree of solvation also affect chromatographic behavior. The selectivity series observed in ion chromatography seems to be best explained by the interplay of two components: electrostatic attraction (ES) and the enforced-pairing (EP) that is brought about by hydrophobic attraction and by water-enforced ion pairing. Selectivity in ion-pair chromatography (IPC) and in zwitterion ion chromatography (ZIC) is affected by both the mobile phase cation and anion. This leads to elution orders for anions that are different from conventional ion-exchange chromatography (IC) of anions where cations are excluded from the stationary phase and have little effect on a separation. The elution order of anions in ZIC is similar to that in IC except for small anions of 2-charge, which are retained more weakly in ZIC. A unique advantage of ZIC is that sample ions can be eluted as ion pairs with pure water as the eluent and a conductivity detector. The mechanism for separation of anions on a zwitterionic stationary phase has been a subject for considerable debate. The available facts point strongly to a partitioning mechanism or a mixed mechanism in which partitioning is dominant with a weaker ion-exchange component.

Preparation and applications of weak acid cation exchanger based on monodisperse poly(ethylvinylbenzene-co-divinylbezene) beads.

New technology is reported here for the synthesis of an effective weak acid-cation exchanger for ion chromatography. Monodisperse macroporous poly(ethylvinylbenzene-co-divinylbezene) (PEVB-DVB) beads of 5 microm diameter were prepared by a two-step swelling and polymerization method. Then carboxyl groups were introduced by polymerization of maleic anhydride with unreacted vinyl groups on the resin beads, followed by hydrolysis of the maleic anhydride groups. A column packed with the carboxylate beads was used to separate alkali and alkaline earth metal ions in a single isocratic run. Separations were found to be better than those with similar resin particles that are simply coated with maleic acid. The columns containing the new particles were 100% compatible with solvents commonly used for HPLC. Additionally, the prepared column was stable and could be used for a long time in a wide range of pH. The column gave good resolution, low detection limits and good repeatability for the separation of common cations. Satisfactory results were also obtained for separations of organic amines and of common cations in rainwater.

Multiplexed colorimetric solid-phase extraction: determination of silver(I), nickel(II), and sample Ph.

This paper reports the design and ground-based testing of a multiplexed colorimetric solid-phase extraction (MC-SPE) platform for the rapid determination of multiple water quality parameters in a simple set of operational steps. Colorimetric solid-phase extraction (C-SPE) is an analytical platform that combines impregnated colorimetric reagents on a solid-phase extraction membrane and diffuse reflectance spectroscopy to quantify trace analytes in water. In extending C-SPE to MC-SPE, a filter holder that incorporates discrete analysis channels and a jig that facilitates the concurrent operation of multiple syringes have been designed, enabling the simultaneous determination of three different measures of water quality. Separate, single-parameter membranes, placed in a readout cartridge create unique, parameter-specific addresses at the exit of each channel. Following sample exposure, the diffuse reflectance spectrum of each address is collected serially and the Kubelka-Munk function is used to quantify each water quality parameter via calibration curves. Performance evaluations of the MC-SPE platform were conducted using sample pH, silver(I), and nickel(II). Determinations of silver(I) (0.05-0.5 ppm) and nickel(II) (1.8-5.0 ppm) follow established C-SPE methods on reversed-phase extraction membranes using 5-(p-dimethylaminobenzylidene)rhodanine and dimethylglyoxime, respectively, as colorimetric reagents. Sample pH (2.5-5.0) is measured using an anion-exchange membrane impregnated with fluorescein. These determinations require approximately 120 s to complete using a total sample volume of 3.0 mL. The extension of MC-SPE to the determination of a greater number of analytes and its potential application to space and earth-bound monitoring needs are briefly discussed.

Early milestones in the development of ion-exchange chromatography: a personal account.

Ion chromatography as we know it today was built on a foundation of knowledge accumulated over a period of many years. Here, we review some of the outstanding earlier achievements in ion-exchange chromatography. Beginning about 1947. Spedding and Powell at Iowa State published a series of papers describing practical methods for preparative separation of the rare earths by displacement ion-exchange chromatography. The same group then demonstrated the ion-exchange separation of 14N and 15N isotopes in ammonia. Beginning in the 1950s. Kraus and Nelson at Oak Ridge published numerous analytical methods for metal ions based on separation of their chloride, fluoride, nitrate or sulfate complexes by anion chromatography. In the period from about 1960 to 1980 many clever chromatographic methods for metal ion separations were reported by researchers throughout the world and automatic in-line detection was gradually introduced. A truly innovative method by Small, Stevens and Bauman at Dow Chemical Co. marked the birth of modern ion chromatography. Anions, as well as cations, could now be separated quickly and conveniently by a system of suppressed conductivity detection. A method for anion chromatography with non-suppressed conductivity detection was published by Gjerde et al. in 1979. This was followed by a similar method for cation chromatography in 1980. Ion chromatography as we know it today did not just happen. It was built on a solid foundation of knowledge that has accumulated over a period of many years. Revisiting the older ion-exchange chromatography serves not only to pay tribute to some remarkable accomplishments, but it can also be a learning experience. Trends and ideas in science tend to run in repeating cycles. Thus, an awareness of older work may provide inspiration for new research using improved contemporary technology. Selection of milestones is a rather personal matter. I chose to write about subjects of which I came to have a firsthand knowledge during my career. The topics selected are in roughly chronological order and cover the period from about 1945 to 1980. An effort has been made to explain the chemical principles as well as to recount the major accomplishments of the various research projects.

Capillary electrophoresis of organic cations at high salt concentrations.

At concentrations of 100 mM or higher the chemical nature of both the cation and anion in the background electrolyte (BGE) can be varied to manipulate the migration times of protonated aniline cations. Significant differences were noted with Li+, Na+ and K+ for capillary electrophoretic runs carried out at pH 3. However, much greater differences in migration times were observed at acidic pH values when the BGE contained protonated cations of aliphatic amines. Analyte migration became progressively slower in the series: methylamine, diethylamine, diethylamino ethanol and triethylamine. A major part of this effect was attributed to an opposing electroosmotic flow (EOF) resulting from a positively-charged coating of the capillary surface with the amine cations in the BGE via a dynamic equilibrium. The amine cations also interact in solution with the analyte ions to reduce their electrophoretic mobilities. Migration times of anilines could be varied systematically over a broad range according to the BGE amine cation selected. Excellent separations of seven closely-related anilines were obtained with the new system.

Separation of anions by ion-interaction chromatography with a novel cationic/zwitterionic eluent.

Inorganic and organic anions can be separated on an ordinary silica C18 column using a mobile phase containing tetrabutylammonium hydroxide (TBAH) and an aminosulfonic acid zwitterion reagent (MOPS). The pH of this eluent is close to 7 and the background conductivity is about 50 microS, which is low enough to permit anion analyte detection by direct conductivity. Linear calibration curves were obtained for the six anions studied and detection limits ranged from 0.075 to 0.15 mg/l (ppm) for the five inorganic anions. The method was applied to the determination of water-soluble anions in aerosol samples at concentrations as low as 0.3 mg/l.

Modification of ion chromatographic separations by ionic and nonionic surfactants.

New findings are reported on simple ways to modify an ordinary HPLC column to obtain efficient ion chromatographic (IC) separations. Permanently coating a column with an ionic surfactant is known to produce an effective column for IC. We now show that incorporation of a nonionic surfactant in the coating, or coating in separate layers, results in a dramatic reduction of ion retention times and gives sharper peaks. Dynamic coating by incorporating a small amount of an alcohol, diol or zwitterion in the aqueous mobile phase permits good separations of alkanecarboxylic acids. A mobile phase containing a quaternary ammonium cation and a zwitterion anion provides excellent separations of common anions on a silica C18 column. An aqueous eluent containing a mixture of a zwitterion 4-(2-hydroxyethyl) acid and methanesulfonic acid can be used in conjunction with a standard cation exchange column. After passing through a membrane suppressor, the mobile phase has a slightly acidic pH, permitting divalent transition metal ions (as well as others) to be detected by conductivity.

Rapid determination of ions by combined solid-phase extraction--diffuse reflectance spectroscopy.

We introduce colorimetric solid-phase extraction (C-SPE) for the rapid determination of selected ions. This new technique links the exhaustive concentration of an analyte by SPE onto a membrane disk surface for quantitative measurement with a hand-held diffuse reflectance spectrometer. The concentration/measurement procedure is complete in approximately 1 min and can be performed almost anywhere. This method has been used to monitor iodine and iodide in spacecraft water in the 0.1-5.0 ppm range and silver(I) in the range of 5.0-1000 microg/l. Applications to the trace analysis of copper(II), nickel(II), iron(III) and chromium(VI) are described. Studies on the mechanism of extraction showed that impregnation of the disk with a surfactant as well as a complexing reagent results in uptake of additional water, which markedly improves the extraction efficiency.

Ion-exclusion chromatography with conductimetric detection of aliphatic carboxylic acids on a weakly acidic cation-exchange resin by elution with benzoic acid-beta-cyclodextrin.

In this study, an aqueous solution consisting of benzoic acid with low background conductivity and beta-cyclodextrin (beta-CD) of hydrophilic nature and the inclusion effect to benzoic acid were used as eluent for the ion-exclusion chromatographic separation of aliphatic carboxylic acids with different pKa values and hydrophobicity on a polymethacrylate-based weakly acidic cation-exchange resin in the H+ form. With increasing concentration of beta-cyclodextrin in the eluent, the retention times of the carboxylic acids decreased due to the increased hydrophilicity of the polymethacrylate-based cation-exchange resin surface from the adsorption of OH groups of beta-cyclodextrin. Moreover, the eluent background conductivity decreased with increasing concentration of beta-cyclodextrin in 1 mM benzoic acid, which could result in higher sensitivity for conductimetric detection. The ion-exclusion chromatographic separation of carboxylic acids with high resolution and sensitivity was accomplished successfully by elution with a 1 mM benzoic acid-10 mM cyclodextrin solution without chemical suppression.

The role of organic solvents in the separation of nonionic compounds by capillary electrophoresis.

Although nonionic compounds can be separated by micellar electrokinetic chromatography (MEKC), application of this technique is restricted by a somewhat limited elution range. Incorporation of organic solvents in the background electrolyte (BGE) greatly extends the scope of MEKC and provides a major variable in optimizing the separation of neutral analytes. This paper provides a systematic review of the principles and scope of the separation of neutral analytes by capillary electrophoresis (CE) in organic-aqueous solution. The methods surveyed include those that use tetraalkylammonium salts, dioctyl sulfosuccinate, lauryl poly(oxyethylene) sulfate. Polyaromatic hydrocarbon (PAH) compounds can be separated using sodium hexadecyl sulfate in 70% methanol (30% aqueous) to 100% methanol.

Ion chromatography on reversed-phase materials coated with mixed cationic and nonionic surfactants.

Columns suitable for use in anion chromatography can be prepared by coating a packed reversed-phase HPLC column (C18 silica or polystyrene particles) with a cationic surfactant. The efficiency is improved dramatically by first coating the column with a nonionic surfactant and then subsequently with the cationic surfactant. The thickness of the first coated layer as well as the chemical structure of the surfactant have a major effect on the column performance. Actual separations are included to demonstrate the convenience and practical use of the coated columns. Using this approach, columns with 12,900 theoretical plates for the 15-cm column (or 86,000 plates/m) were produced, giving well shaped peaks with an average asymmetry factor of 1.09. The coated layers were found to be stable, giving retention times with an average relative standard deviation of 1.6% for 12 consecutive runs.