Simple and selective assay of 4-hydroxymephenytoin in human urine using solid-phase extraction and high-performance liquid chromatography with electrochemical detection and its preliminary application to phenotyping test

A simple and selective HPLC method for the determination of 4-hydroxymephenytoin (4-OH-M) in human urine, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite, has been developed. After acid hydrolysis of urine, 4-OH-M and the internal standard (I.S.), 5-hydroxy-1-tetralone, were extracted from urine by means of a Bond Elut Certify LRC column. The extracts were chromatographed on a reversed-phase μBondapak C₁₈ column using methanol-50 mM KH₂PO₄ (pH 4.0) (30:70, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Electrochemical detection at applied potential of 800 mV resulted in a limit of quantitation of 0.76 μg/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. The present method was applied to the phenotyping test in thirteen Japanese healthy volunteers who recieved an oral 10-mg racemic mephenytoin. The phenotypes determined by the present method were found to be in agreement with those obtained with the reported customary assay based on gas chromatography.     

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.     

Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease

Analytical methods are described for the selective, rapid and sensitive determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The glucuronide and sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-apomorphine a 10 μm Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min⁻¹ the total run time is ca. 15 min. The detection limits are 0.3 and 0.6 ng ml⁻¹ for R- and S- apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml⁻¹ for plasma samples, and <4% in the concentration range of 40-400 ng ml⁻¹ for urine samples. For the assay of apomorphine, apocodeine and isoapocodeine, a 5 μm C₁₈ column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a flow-rate of 0.8 ml min⁻¹ the total run time is ca. 14 min. The detection limits of this assay are 1.0 ng ml⁻¹ for apomorphine and 2.5 ng ml⁻¹ for both apocodeine and isoapocodeine (signal-to-noise ratio 3). The inter-assay variations are 5% in the concentration range of 5-40 ng ml⁻¹ for plasma samples and 7% in the concentration range of 50-500 ng ml⁻¹ for urine samples. The glucuronic acid and sulfate conjugates of the various compounds are hydrolysed by incubation of the samples with β-glucuronidase and sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of apomorphine, apocodeine and isoapocodeine occurred during the incubation. A pharmacokinetic study of apomorphine, following the intravenous infusion of 30 μg kg⁻¹ for 15 min in a patient with Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent drug and the appearance of apomorphine plus metabolites in urine could be determined.     

Auxin levels in single half nodes ofAvena fatua estimated using high performance liquid chromatography with coulometric detection

A high performance liquid chromatography (HPLC) based micro-method for estimation of indole-3-acetic acid (IAA) levels in single half nodes from the flowering stalks ofAvena fatua has been developed; this features a dual electrode coulometric electrochemical detector operating at a detection limit of c. 2 pg. Samples were prepared by solvent partitioning and preliminary fractionation with C¹⁸ Sep-Pak cartridges. Two stages of reversed phase ion pair HPLC were employed; the first was gradient elution with fluorescent detection, the second, isocratic elution with coulometric detection. The lower limit for estimation of IAA levels in purified extracts was c. 5 pg.     

Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography

An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α₁-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.     

Application of the dual-cell coulometric detector: a method for assaying monoamines and their metabolites

A new HPLC assay technique for monoamines and their metabolites, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite through which the samples flow, is described in comparison with a classical amperometric detector equipped with a glassy carbon electrode. Different potentials can be applied at each cell of the coulometric detector to improve sample resolution and detection sensitivity. The signal-to-noise ratio (s/n) calculated in similar conditions was 10 times lower for the coulometric detector than for the amperometric one. The dual-coulometric detector does not undergo daily decay or variation, and needs no particular care or preparation. It is therefore possible to achieve stable routine sensitivity in a range of 10 fmol. This new technique has been applied for assaying monoamines and their precursors and metabolites by direct injection of clear supernatant after centrifugation and for determination of catecholamine turnover in rat pineal gland and neuro- and adenohypophysis in samples purified by Al2O3 adsorption.     

High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine

A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10⁻⁷M.     

Auxin levels in single half nodes ofAvena fatua estimated using high performance liquid chromatography with coulometric detection

A high performance liquid chromatography (HPLC) based micro-method for estimation of indole-3-acetic acid (IAA) levels in single half nodes from the flowering stalks ofAvena fatua has been developed; this features a dual electrode coulometric electrochemical detector operating at a detection limit of c. 2 pg. Samples were prepared by solvent partitioning and preliminary fractionation with C¹⁸ Sep-Pak cartridges. Two stages of reversed phase ion pair HPLC were employed; the first was gradient elution with fluorescent detection, the second, isocratic elution with coulometric detection. The lower limit for estimation of IAA levels in purified extracts was c. 5 pg.     

Structure Elucidation of the Metabolites of 2', 3', 5'-Tri-O-Acetyl-N 6-(3-Hydroxyphenyl) Adenosine in Rat Urine by HPLC-DAD,ESI-MS and Off-Line Microprobe NMR

2'', 3'', 5''-tri-O-acetyl-N₆-(3-hydroxyphenyl) adenosine (also known as WS070117) is a new adenosine analog that displays anti-hyperlipidemic activity both in vitro and in vivo experiments as shown in many preliminary studies. Due to its new structure, little is known about the metabolism of WS070117. Hence, the in vivo metabolites of WS070117 in rat urine following oral administration were investigated. Identification of the metabolites was conducted using the combination of high-performance liquid chromatography (HPLC) coupled with diode array detector (DAD), ion trap electrospray ionization-mass spectrometry (ESI-MS), and off-line microprobe nuclear magnetic resonance (NMR) measurements. Seven metabolites were obtained as pure compounds at the sub-milligram to milligram levels. Results of structure elucidation unambiguously revealed that the phase I metabolite, N₆-(3-hydroxyphenyl) adenosine (M8), was a hydrolysate of WS070117 by hydrolysis on the three ester groups. N₆-(3-hydr-oxyphenyl) adenine (M7), also one of the phase I metabolites, was the derivative of M8 by the loss of ribofuranose. In addition to two phase I metabolites, there were five phase II metabolites of WS070117 found in rat urine. 8-hydroxy-N₆-(3-hydroxy-phenyl) adenosine (M6) was the product of M7 by hydrolysis at position 8. The other four were elucidated to be N₆-(3-O-β-D-glucuronyphenyl) adenine (M2), N₈-hydroxy-N₆-(3-O-sulfophenyl) adenine (M3), N₆-(3-O-β-D-glucuronyphenyl) adenosine (M4), and N₆-(3-O- sulfophenyl) adenosine (M5). Phase II metabolic pathways were proven to consist of hydroxylation, glucuronidation and sulfation. This study provides new and valuable information on the metabolism of WS070117, and also demonstrates the HPLC/MS/off-line microprobe NMR approach as a robust means for rapid identification of metabolites.     

Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

Two ferrocene-labelled analogues of dTTP, 5-(3-ferrocenecarboxamidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc1-dUTP) and 5-(3-ferroceneacet-amidopropenyl-1) 2′-deoxyuridine 5′-triphosphate (Fc2-dUTP) have been produced to demonstrate the incorporation of redox labels into DNA by polymerases. Cyclic voltammetry indicates that the ferrocenyl moieties display reversible redox behaviour in aqueous buffer with E_1/2 values of 398 (Fc1-dUTP) and 260 mV (Fc2-dUTP) versus Ag/AgCl. Primer extension by the proofreading enzymes Klenow fragment and T4 DNA polymerase shows that Fc1-dUTP is efficiently incorporated into DNA during synthesis, including incorporation of two successive modified nucleotides. Production of a 998 bp amplicon by Tth DNA polymerase demonstrates that Fc1-dUTP is also a satisfactory substrate for PCR. Despite its structural similarity, Fc2-dUTP acts predominantly as a terminator with the polymerases employed here. UV melting analysis of a 37mer duplex containing five Fc1-dU residues reveals that the labelled nucleotide introduces only a modest helix destabilisation, with T_m = 71 versus 75°C for the corresponding natural construct. Modified DNA is detected at femtomole levels using a HPLC system with a coulometric detector. The availability of simple and effective enzymatic labelling strategies should promote the further development of electrochemical detection in nucleic acid analysis.     

Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

Determination of piretanide and furosemide in pharmaceuticals and human urine by high-performance liquid chromatography with amperometric detection

A high-performance liquid chromatographic method with electrochemical detection (ED) has been developed for the determination of two diuretics: 4-phenoxy-3-(1-pyrrolidinyl)-5-sulfamoylbenzoic acid (piretanide) and 4-chloro-2-furfurylamino-5-sulfamoylbenzoic acid (furosemide). The chromatographic separation was performed on a μBondapak C₁₈ column with a mobile phase of acetonitrile-water (40:60) containing 5 mM KH₂PO₄/K₂HPO₄ and with a flow-rate of 1 ml/min (69 bar). The temperature was optimized at 30 ± 0.2°C. The amperometric detector equipped with a glassy carbon electrode was operated at + 1200 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds in two concentration ranges (ppm and ppb), obtaining a reproducibility in terms of relative standard deviations lower than 1% for within-day and 4% for day-to-day and determination limits of 15 ppb for both compounds. Recoveries greater than 90% were obtained for spiked urine samples, using a liquid-liquid extraction method in the sample clean-up procedure. The LC-ED method was applied to commercially available pharmaceuticals (Seguril, furosemide 40 mg, and Perbilén, piretanide 6 mg) and urine samples obtained from healthy volunteers and hypertensive patients.     

Determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de[1,4] benzoxazine-6-carboxylic acid hemihydrate,DV-7751a,in human serum and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl)]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido [1,2,3-de][1,4]benzoxazine-6-carboxylic acid hemihydrate (DV-7751a, I) in human serum and urine has been developed. Compound I and the internal standard were extracted from serum and urine by means of Bond Elut C₈ LRC column. The extracts were chromatographed on a reversed-phase Inertsil ODS-2 column using tetrahydrofuran-50 mM KH₂PO₄ (pH 2)-1 M ammonium acetate (19:81:1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Fluorescence detection at an excitation wavelength of 305 nm and an emission wavelength of 530 nm resulted in a limit of quantitation of 0.0098 μg/ml for serum and 0.098 μg/ml for urine. The method showed satisfactory sensitivity, precision, accuracy, recovery and selectivity. Stability studies showed that I was stable in serum and urine for at least 1 month at −20°C and for at least 48 h at room temperature.     

Electrochemical and Thermodynamic Properties of Ln(III) (Ln?=?Eu,Sm, Dy,Nd) in 1-Butyl-3-Methylimidazolium Bromide Ionic Liquid

The electrochemical behavior and thermodynamic properties of Ln(III) (Ln = Eu, Sm, Dy, Nd) were studied in 1-butyl-3-methylimidazolium bromide ionic liquid (BmimBr) at a glassy carbon (GC) electrode in the range of 293–338 K. The electrode reaction of Eu(III) was found to be quasi-reversible by the cyclic voltammetry, the reactions of the other three lanthanide ions were regarded as irreversible systems. An increase of the current intensity was obtained with the temperature increase. At 293 K, the cathodic peak potentials of −0.893 V (Eu(III)), −0.596 V (Sm(III)), −0.637 V (Dy(III)) and −0.641 V (Nd(III)) were found, respectively, to be assigned to the reduction of Ln(III) to Ln(II). The diffusion coefficients (D _o), the transfer coefficients (α) of Ln(III) (Ln = Eu, Sm, Dy, Nd) and the charge transfer rate constants (k _s) of Eu(III) were estimated. The apparent standard potential (E ⁰*) and the thermodynamic properties of the reduction of Eu(III) to Eu(II) were also investigated.     

Differential pulse amperometric detection of drugs in plasma using a dropping mercury electrode as a high-performance liquid chromatographic detector

High-performance liquid chromatographic separation prior to reductive electrochemical determination at the dropping mercury electrode imparts specificity and sensitivity not attainable by conventional polarographic analysis of drugs and their metabolites. The utility of this novel approach is demonstrated by the analysis of chlordiazepoxide and its N-desmethyl metabolite in plasma which previously required thin-layer chromatographic separation prior to polarographic measurement. A mobile phase of methanol—isopropanol—0.0075 M acetate buffer, pH 3.5 (53:5:42), is used with the detector operated in the differential pulse mode at E_p = ?0.820 V vs. Ag/AgCl. The response was linear (r = 0.998) in the concentration range of 0.05–2.0 μg/ml plasma for each component. The minimum detectability for each component under these conditions is 5.0 ng injected at a current range of 0.5 μA full scale. Techniques for oxygen removal and hydrodynamic considerations for the pumping system are presented.     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (T_m(L)) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T_m(L). The average T_m(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.     

High-performance liquid chromatography with amperometric detection applied to the screening of β-blockers in human urine

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of six β-blockers; atenolol, nadolol, timolol, metoprolol, oxprenolol, and alprenolol.The chromatographic separation was performed using a μBondapack C₁₈ column, a mobile phase of acetonitrile-water (40:60), containing 5 mM KH₂PO₄/K₂HPO₄ proved to be optimal at a 1.3 ml/min flow-rate, and a pH of 6.5. The temperature was optimized at 30±0.2°C. The amperometric detector, equipped with a glassy carbon electrode, was operated at 1300 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/ml), obtaining relative standard deviations lower than 5% at ppm levels and lower than 10% at ppb levels, and quantitation limits ranging from 15 ppb to 500 ppb.The method was applied to the screening of β-blockers in spiked urine samples, with a total elution time lower than 12 min, obtaining the best recoveries for timolol and metoprolol (never greater than 93%). These recoveries together with the low limits of quantitation achieved, allows its application to doping analysis in human urine.     

Structural Disorder of Folded Proteins: Isotope-Edited 2D IR Spectroscopy and Markov State Modeling

The conformational heterogeneity of the N-terminal domain of the ribosomal protein L9 (NTL9_1-39) in its folded state is investigated using isotope-edited two-dimensional infrared spectroscopy. Backbone carbonyls are isotope-labeled (¹³C=¹⁸O) at five selected positions (V3, V9, V9G13, G16, and G24) to provide a set of localized spectroscopic probes of the structure and solvent exposure at these positions. Structural interpretation of the amide I line shapes is enabled by spectral simulations carried out on structures extracted from a recent Markov state model. The V3 label spectrum indicates that the β-sheet contacts between strands I and II are well folded with minimal disorder. The V9 and V9G13 label spectra, which directly probe the hydrogen-bond contacts across the β-turn, show significant disorder, indicating that molecular dynamics simulations tend to overstabilize ideally folded β-turn structures in NTL9_1-39. In addition, G24-label spectra provide evidence for a partially disordered α-helix backbone that participates in hydrogen bonding with the surrounding water.     

Infrared spectroscopic studies of aqueous solutions of dioxouranium(VI) and its hydrolysed products and of in situ electro-generated dioxouranium(V)

Aqueous solutions of dioxouranium(VI) (pH range 0 to 4) give rise to bands at 954 and 938 cm⁻¹ attributable to the v₃(MO₂) stretching modes of the UO₂²⁺ and (UO₂)₂(OH)_2²⁺ cations, respectively. A shoulder at 916 cm⁻¹ is assigned to the v₃(MO₂) mode of hydrolysed dioxouranium(VI) species of higher nuclearity. Infrared spectro-electrochemical studies using a thin-layer reflection-absorption cell have facilitated the study of the reduction of aqueous solutions of dioxouranium(VI) to yield dioxouranium(V) which may be further reduced to uranium(IV). The electrogeneration of dioxouranium(V) is monitored by following the increase in intensity of a band at 914 cm⁻¹ which is present in the spectra at potentials between −0.2 and −0.8 V. The dioxouranium(V) species is predominantly in the form UO₂⁺, which may be in solution or incorporated into an insoluble phase of uranium oxides which deposit onto the working electrode. The U^VO bond length is estimated to be 1.76 Å, 0.03 Å longer than the U^VIO bond in aqueous solution. The maximum concentration of UO₂⁺able to be achieved is highly dependent on the pH and is optimum at pH 3.4. Changes in the pH of the solution under study can be monitored by infrared spectroscopy during the course of the reduction by determining the relative concentrations of hydrolysed dioxouranium(VI) species.