A simple LC-MS method for the determination of iohexol and iothalamate in serum, using ioversol as an internal standard.

BACKGROUND: Chronic kidney disease (CKD) is diagnosed and explored through the determination of the glomerular filtration rate (GFR). Our goal was to develop a simple LC-MS method for the determination in serum of 2 popular GFR markers, contrast agents iohexol and iothalamate, for routine use and comparison studies between the two markers. A similar contrast agent, ioversol, was used as an internal standard and the method underwent a rigorous validation protocol based on ß-expectation tolerance intervals. METHODS: We adapted the HPLC-UV method from Cavalier et al. to our LC-MS system. Data treatment for the validation was performed using Multiquant 3.0 (Sciex, Framingham, MA, USA) and e.noval 3.0 software (Arlenda, Liège, Belgium). RESULTS: According to the validation results our method will give accurate and reliable results for concentrations ranging from 6.8 to 250µg/ml for iohexol and 6.15µg/ml to 250µg/ml for iothalamate. In our practice these intervals are sufficient to determine both compounds in most patient samples. Samples with higher detected concentrations can always be diluted into range. CONCLUSION: With its internal standard and extensive validation, our method is now ready for routine and clinical research use.

Determination of iohexol and iothalamate in serum and urine by capillary electrophoresis.

Estimation of glomerular filtration rate (eGFR) is essential to assess kidney function. Iodine-containing contrast agents detection by HPLC has been proposed as a safe alternative for inulin or radioactive compounds. However, HPLC is a time-consuming and labor-intensive method. The aim of this study was to develop an assay for iohexol and iothalamate using capillary electrophoresis. Iohexol and iothalamate were directly analyzed by CE in serum and urine, using photometric detection (246 nm). Serum peak height was proportional to iohexol and iothalamate concentrations. Detection limits for iohexol and iothalamate were 10 and 5 mg/L. Limits of quantification were 13.0 and 15.0 mg/L. Within-run CVs were 4.9 and 6.5%; between-run CVs 3.1-9.9% and 3.8-13.7%. A good correlation was observed between CE and HPLC: y = 1.1703x + 5.017 (iohexol) and y = 0.7807x + 11.01 (iothalamate; (y = concentration obtained by CE [mg/L], x = concentration obtained by HPLC [mg/L]). In addition, CE allowed to determine urinary iohexol concentration. Although the detection limit for CE was higher than for HPLC, CE can still be used for eGFR determination. Advantages of this high-throughput method are the absence of sample pretreatment and a minimal sample volume requirement.

Discordance Between Iothalamate and Iohexol Urinary Clearances.

BACKGROUND: Iothalamate and iohexol are contrast agents that have supplanted inulin for the measurement of glomerular filtration rate (GFR) in clinical practice. Previous studies have noted possible differences in renal handling of these 2 agents, but clarity about the differences has been lacking. STUDY DESIGN: Study of diagnostic test accuracy. SETTING & PARTICIPANTS: 150 participants with a wide range of GFRs were studied in an outpatient clinical laboratory facility. INDEX TESTS: Simultaneous urinary clearances of iothalamate, iohexol, and creatinine. REFERENCE TEST: None. OUTCOME: Relative differences between the urinary clearances. Iohexol and iothalamate in plasma and urine were assayed concurrently by a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. RESULTS: Mean iohexol, iothalamate, and creatinine clearances were 52±28 (SD), 60±34, and 74±40 mL/min/1.73 m(2), respectively. The proportional bias of iohexol to iothalamate urinary clearance was 0.85 (95% CI, 0.83-0.88) and was proportional across the GFR range. The mean proportional bias of iohexol clearance compared with creatinine clearance is 1.27 (95% CI, 1.20-1.34), whereas that of iothalamate clearance compared with creatinine clearance is 1.09 (95% CI, 1.03-1.15). LIMITATIONS: Lack of reference standard. CONCLUSIONS: This study reveals a significant and consistent difference between urinary clearances of iothalamate and iohexol. Comparison of studies reporting renal clearance measurements using iohexol versus iothalamate must account for this observed bias.

Cystatin C enhances glomerular filtration rate estimating equations in kidney transplant recipients.

BACKGROUND: The glomerular filtration rate (GFR) estimating equation incorporating both cystatin C and creatinine perform better than those using creatinine or cystatin C alone in patients with reduced GFR. Whether this equation performs well in kidney transplant recipients cross-sectionally, and more importantly, over time has not been addressed. METHODS: We analyzed four GFR estimating equations in participants of the Angiotensin II Blockade for Chronic Allograft Nephropathy Trial (NCT 00067990): Chronic Kidney Disease Epidemiology Collaboration equations based on serum cystatin C and creatinine (eGFR (CKD-EPI-Creat+CysC)), cystatin C alone (eGFR (CKD-EPI-CysC)), creatinine alone (eGFR (CKD-EPI-Creat)) and the Modification of Diet in Renal Disease study equation (eGFR (MDRD)). Iothalamate GFR served as a standard (mGFR). RESULTS: mGFR, serum creatinine, and cystatin C shortly after transplant were 56.1 ± 17.0 ml/min/1.73 m(2), 1.2 ± 0.4 mg/dl, and 1.2 ± 0.3 mg/l respectively. eGFR (CKD-EPI-Creat+CysC) was most precise (R(2) = 0.50) but slightly more biased than eGFR (MDRD); 9.0 ± 12.7 versus 6.4 ± 15.8 ml/min/1.73 m(2), respectively. This improved precision was most evident in recipients with mGFR >60 ml/min/1.73 m(2). For relative accuracy, eGFR (MDRD) and eGFR (CKD-EPI-Creat+CysC) had the highest percentage of estimates falling within 30% of mGFR; 75.8 and 68.9%, respectively. Longitudinally, equations incorporating cystatin C most closely paralleled the change in mGFR. CONCLUSION: eGFR (CKD-EPI-Creat+CysC) is more precise and reflects GFR change over time reasonably well. eGFR (MDRD) had superior performance in recipients with mGFR between 30 and 60 ml/min/1.73 m(2).

Use of dried plasma spots for the quantification of iothalamate in clinical studies.

BACKGROUND AND OBJECTIVES: Although iothalamate clearances have been widely used to measure GFR, the need for transportation of plasma samples under refrigerated conditions obviates its use in resource-poor situations. Spots of blood or plasma dried on filter paper may provide a solution. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Using a validated HPLC technique, iothalamate in dried blood spots of different hematocrits was measured. GFR was measured over 5 hours in 10 subjects with CKD using dried plasma spots and standard methods. RESULTS: Lower hematocrit produced greater area of blood spreading and lowered the recovery of iothalamate from dried blood spots. However, the relationship between iothalamate concentrations in dried plasma spots and plasma showed a regression slope of 0.95 (95% confidence interval=0.92-0.98, P<0.001). Bland-Altman plot of paired sample points (n=116) showed a bias of -4 µg/ml and limits of agreement of -38 to +30 µg/ml. The relationship between GFRs using dried plasma spots and plasma methods also showed an excellent relationship (slope of 0.95, 95% confidence interval=0.82-1.17). Bland-Altman plot of paired GFRs showed a bias of 2 ml/min, with limits of agreement of -6 to +10 ml/min. Precision was generally between 5% and 10%, and accuracy was within 5%. CONCLUSIONS: Although dried blood spots are unsuitable for studies among those patients with very low hematocrit, dried plasma spots correct for this limitation, and this small pilot study shows that it is a reasonably reliable method for quantifying iothalamate and subsequently, determining GFR.

Development of a liquid chromatography tandem mass spectrometry method for iothalamate measurement to assess renal function for potential kidney donation.

BACKGROUND: Chronic kidney disease often goes undetected due to the insensitivity of current methods to accurately assess glomerular filtration rate (GFR) in early stages of renal dysfunction. The clearance of exogenously introduced iothalamate, a commonly used radiopaque agent, is an alternative to inulin clearance for the assessment of renal function and its use in calculating GFR can serve as a screening tool for kidney transplant donors. METHODS: A method was developed to measure iothalamate in plasma and urine samples by HPLC combined with electrospray positive ionization tandem mass spectrometry (MS/MS). Iothalamate is isolated from plasma by methanol extraction and urine using a quick-spin filtration approach, then monitored by multiple reaction monitoring using the hydrogen adduct mass transitions. Iohexol was used as an internal standard. RESULTS: Iothalamate was measured within an analytical run time of 5 min, with a lower limit of quantification of 18.75 ng/ml. The intraassay and interassay variations of the plasma and urine iothalamate assays were both <9%. Recovery from plasma and urine samples ranged from 93.6% to 104.1%. GFR was calculated using the patient's urine flow rate and plasma and urine iothalamate values. Linear correlations tested by LC-MS/MS and an accepted capillary electrophoresis (CE) assay showed similar results (GFR, r=0.92, Sy/x=10.3). CONCLUSIONS: We developed and validated an LC-MS/MS method for quantitating iothalamate in plasma and urine to calculate GFR used for screening potential kidney donors in our hospital system. A less sensitive mass spectrometry system does not sacrifice analytical or clinical sensitivity for measuring GFR.

Estimation of glomerular filtration rate by using serum cystatin C and serum creatinine concentrations in patients with human immunodeficiency virus.

STUDY OBJECTIVE: To compare the predictive performance of four equations for estimating glomerular filtration rate (GFR) relative to the gold standard measurement, iothalamate clearance, in patients with human immunodeficiency virus (HIV) who have various degrees of kidney function. DESIGN: Prospective, cross-sectional analysis. SETTING: General clinical research center. PATIENTS: Twenty-two adult (mean age 51 yrs) HIV-positive patients with various degrees of stable kidney function and with lean body mass considered normal for a well-nourished person. INTERVENTION: Patients were administered a single dose of intravenous iothalamate 456 mg as a rapid infusion over 3 minutes, 1 hour after an oral fluid load of 600 ml of caffeine-free, sugar-free liquids. MEASUREMENTS AND MAIN RESULTS: Serial blood and urine samples were obtained for determination of measured GFR. Estimated GFR values were calculated by using four equations: the Cockcroft-Gault equation, the simplified Modification of Diet in Renal Disease Study (MDRD) equation, an equation that incorporates serum creatinine and cystatin C concentrations, and an equation incorporating only serum cystatin C concentration. The predictive performance of the equations was determined by comparing the bias, accuracy, and precision of the estimates with the measured values. Body composition was determined by dual-energy x-ray absorptiometry. The four predictive equations underestimated the measured GFR obtained by the iothalamate method, but the differences were not statistically significant. The MDRD equation and the equation that included both serum cystatin C and creatinine concentrations, as well as age, sex, and race, provided the least bias, most precision, and best accuracy in estimating the measured GFR. CONCLUSION: The MDRD equation and the equation that included both serum cystatin C and creatinine concentrations appear to provide accurate, precise, and relatively unbiased estimates of GFR in patients with HIV. Larger studies are needed that include patients with muscle wasting and lipodystrophy in order to validate these preliminary observations and the effects of body composition on the predictability of GFR with use of these equations.

Iothalamate quantification by tandem mass spectrometry to measure glomerular filtration rate.

BACKGROUND: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. METHODS: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8-487.0, 614.8-456.0, 614.8-361.1, and 614.8-177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. RESULTS: Mean recovery was 90%-110% in both urine and plasma matrices. Imprecision was

Quantitation of iothalamate in urine and plasma using liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS).

The following chapter describes a method to measure iothalamate in plasma and urine samples using high performance liquid chromatography combined with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Methanol and water are spiked with the internal standard (IS) iohexol. Iothalamate is isolated from plasma after IS spiked methanol extraction and from urine by IS spiked water addition and quick-spin filtration. The plasma extractions are dried under a stream of nitrogen. The residue is reconstituted in ammonium acetate-formic acid-water. The reconstituted plasma and filtered urine are injected into the HPLC-ESI-MS/MS. Iothalamate and iohexol show similar retention times in plasma and urine. Quantification of iothalamate in the samples is made by multiple reaction monitoring using the hydrogen adduct mass transitions, from a five-point calibration curve.

Rapid analysis of iodinated X-ray contrast media in secondary and tertiary treated wastewater by direct injection liquid chromatography-tandem mass spectrometry.

The iodinated X-ray contrast media are the most widely administered intravascular pharmaceuticals and are known to persist in the aquatic environment. A rapid method using direct injection liquid chromatography-tandem mass spectrometry (DI-LC-MS/MS) has been developed to measure eight ICM. These include iopamidol, iothalamic acid, diatrizoic acid, iohexol, iomeprol, iopromide, plus both ioxaglic acid and iodipamide, which have not previously reported in the literature. The LC-MS/MS fragmentation patterns obtained for each of the compounds are discussed and the fragments lost for each transition are identified. Matrix effects in post-RO water, MQ water, tap water and secondary effluent have also been investigated. The DI-LC-MS/MS method was validated on both secondary and tertiary treated wastewater, and applied to samples from an advanced activated sludge wastewater treatment plant (WWTP) and a water recycling facility using microfiltration (MF) and reverse osmosis (RO) in Perth, Western Australia. As well as providing information of the efficacy for RO to remove specific ICM, these results also represent the first values of ICM published in the literature for Australia.

High performance liquid chromatographic measurement of iothalamate in human serum and urine for evaluation of glomerular filtration rate.

A simple and sensitive HPLC-UV assay was developed for the measurement of iothalamate (IOT) in human serum and urine. Chromatographic separation was achieved using an embedded-carbamate-group bonded RP18 column and mobile phase consisting of 50 mM monobasic sodium phosphate and methanol (90:10, v/v) without the addition of ion-pair reagents. The assay demonstrated a high analytical reliability within the IOT concentration range of 1-150 microg/ml in serum and 25-1500 microg/ml in urine. The relative standard deviations (RSDs) for intra- and inter-day analysis were less than 5.1% in all cases. This method has been used for the evaluation of glomerular filtration rate (GFR) in subjects participating in a phase I clinical trial of a novel antimalarial medicine. The average baseline GFR was 100.41+/-19.99 ml/min/1.73 m(2) in 119 healthy volunteers. The assay may also allow the simultaneous measurements of p-aminohippuric acid (PAH), N-acetyl PAH (aPAH), and IOT with some modification. PAH, IOT, aPAH, and beta-hydroxyethyl-theophylline internal standard peaks appeared approximately at 2.5, 3.7, 5.9, and 11.8 min, respectively, in an isocratic run.

Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.

Analysis of iodinated X-ray contrast agents in water samples by ion chromatography and inductively-coupled plasma mass spectrometry.

In this paper, an analytical method for the determination of six iodinated X-ray contrast agents (amidotrizoic acid, iohexol, iomeprol, iopamidol, iopromide, and ioxitalamic acid), iodide, and iodate in water samples is presented. The method is based on a separation of the analytes by ion chromatography (IC) and a subsequent detection by inductively-coupled plasma mass spectrometry (ICP-MS). The method was optimised with respect to separation conditions (column type and eluent composition) and extensively validated. Without pre-concentration of the samples, limits of detection below 0.2 microg/l could be achieved whereby reproducibility was below 6% for all compounds under investigation.

Simultaneous determination of anions in nanoliter volumes.

BACKGROUND: The study of renal tubular transport requires the ability to accurately measure ion concentrations in samples taken from single tubules. Sample collection and analysis are laborious, so methods allowing determination of multiple ion species in a small volume sample are advantageous. This article describes a method for the simultaneous analysis of anions at physiologic concentrations in nanoliter volumes of tubular fluid. METHOD: The analysis is performed using capillary zone electrophoresis. Diluted samples are moved along a capillary by bulk transport and separated according to charge and size. Peaks corresponding to anions are obtained by ultraviolet (UV) detection; peak area is proportional to ion concentration. RESULTS: The anions chloride, nitrate, citrate, phosphate, and bicarbonate were separated in less than 4 minutes, and iothalamate in less than 5 minutes. Simultaneous quantitative analysis was performed for chloride, phosphate, and bicarbonate, demonstrating detection limits of 12 fmol for chloride, 12 fmol for phosphate, and 72 fmol for bicarbonate. A comparison between this method and a flow-through microfluorimeter analysis of chloride showed good agreement between the two micro-methods. Illustrative data from proximal and distal tubular fluid samples obtained by micropuncture (volume 30-70 nL) are given, as are results from urine samples. RESULTS: Results for chloride, phosphate, and bicarbonate in control material are in close agreement with the certified values, while values in tubular fluid are in accordance with previously published results. CONCLUSION: This method provides a straightforward means of analyzing multiple anions in small volume biological samples.

Ambulatory GFR measurement with cold iothalamate in adults with chronic kidney disease.

BACKGROUND: The purpose of this study is to develop a method that eliminates the use of radioactive agents, reduces the length of inpatient visits, and does not require urine collection for the measurement of glomerular filtration rate (GFR) in adults with chronic kidney diseases (CKD). METHODS: We measured simultaneous urinary and plasma clearance of iothalamate using high-performance liquid chromatography in 30 patients with CKD after a continuous subcutaneous infusion of iothalamate at a rate of 125 microL/h for a 24-hour period. To ascertain whether a steady state was achieved with a 24-hour infusion, in 20 additional patients, we infused iothalamate continuously for 48 hours and measured GFR after 24 and 48 hours. RESULTS: Plasma iothalamate levels and urinary excretion rates obtained after 24- and 48-hour infusions were similar. GFR estimated by plasma clearance (44.4 mL/min; 95% confidence interval [CI], 37.0 to 53.3) was similar to urinary clearance (41.6 mL/min; 95% CI, 34.7 to 50.0) and without bias (average difference, 7%; 95% CI, +18% to -38%; P = 0.866). There also was acceptable agreement between plasma and urinary clearance of iothalamate (coefficient of variation [CV], 19.6% between techniques). Reproducibility of day-to-day plasma continuous infusion clearance technique was less than 12%. There was good agreement (CV, 19.9%) between inulin and renal iothalamate clearances. CONCLUSION: These data show that ambulatory infusion of iothalamate can be used to measure GFR in adults with CKD without the need for urine collections with acceptable precision and without exposure to radioactivity.

Liquid chromatography for iothalamate in biological samples.

We have previously reported an iothalamate assay for the assessment of the glomerular filtration rate (GFR) that required a long column equilibration time and 22 min run time per sample. We now report a simpler assay that requires a run time of only 5.5 min and is more precise and accurate than the earlier technique. The mobile phase consisted of methanol-acetonitrile-50 mM sodium monobasic phosphate (10:5:85, v/v) at pH 4.4, pumped at a rate of 1.5 ml/min on a C(18) reversed-phase column. Samples of plasma and urine were deproteinized with 1 volume of 4% perchloric acid or 9 volumes of 2% perchloric acid, respectively. No internal standard was used. The diode array detection system collected absorbance at 240 nm and the peak height areas of iothalamate were determined. The iothalamate peak appeared at 3.5 min. Detector response was linear over the range tested (10-2000 microg/ml). Within-run precision was <3% for both plasma and urine and accuracy was 96-102%. Between-day precision for plasma and urine analyses were <7%. The recovery of iothalamate in urine and plasma were 102% and 91%, respectively. There was excellent thermal and pH stability of iothalamate. No interference was found with para-amino hippuric acid (PAH) or N-acetyl PAH, which can be simultaneously assayed, if desired.

Iothalamate measured by capillary electrophoresis is a suitable alternative to radiolabeled inulin in renal micropuncture.

BACKGROUND: Inulin remains the gold standard for measurements of fluid reabsorption (Jv) and single nephron glomerular filtration rate (SNGFR) in micropuncture experiments. However, the method used to measure cold inulin in nanoliter samples is time-consuming, while the use of radiolabeled inulin is disadvantaged by possible radioactive contamination, disposal of radioactive material and cost of the isotope. It has been reported that non-radiolabeled iothalamate may be a suitable alternative for estimation of whole kidney GFR. The present study tested whether iothalamate can be used to measure Jv in microperfusion and free-flow micropuncture experiments. METHODS: Superficial loops of Henle (LOH) were perfused from late proximal to early distal tubules with an end-like proximal solution. In the first set of experiments, the perfusate contained both iothalamate (1.9 mmol/L) and 3H-methoxy-inulin (50 microCi. mL(-1)). To test if iothalamate was able to detect changes in Jv, two additional sets of experiments were performed: (1) mannitol (61 mmol/L) was added to the perfusate to partially replace NaCl, a condition known to inhibit Jv; (2) LOH of remnant kidneys were perfused, which in previous experiments we showed to have a higher Jv. Lastly, free-flow micropuncture experiments were performed by infusing iothalamate IV at 18.3 mg. h(-1). Iothalamate analysis in nanoliter samples of renal tubular fluid obtained in vivo was performed by capillary electrophoresis (CE). RESULTS: In the first set of experiments, liquid scintillation counting of 3H-methoxy-inulin versus iothalamate analysis with CE resulted in almost identical calculated perfusion rates (20.4 +/- 0.6 vs. 20.6 +/- 0.7 nL. min(-1), N = 20) and tubular fluid/perfusate ratios (TF/P; 1.35 +/- 0.04 vs. 1.36 +/- 0.04) and thus also Jv (5.17 +/- 0.50 vs. 5.38 +/- 0.59 nL. min(-1)). In the mannitol experiments, iothalamate measurements showed that the addition of mannitol significantly reduced Jv from 4.98 +/- 0.40 (N = 19) to 0.72 +/- 0.58 nL. min(-1) (N = 33; P < 0.0001). Iothalamate determinations by CE were able to detect a significant increase in Jv in LOH of remnant rats perfused at 40 nL. min(-1)[from to 8.40 +/- 0.73 (N = 20) in sham-operated to 17.8 +/- 2.9 nL. min(-1) (N = 6) in remnant animals; P < 0.0001]. In free flow micropuncture experiments the ratio of tubular fluid to plasma iothalamate (TF/P) along the proximal tubule was 1.62 +/- 0.10 (N = 15). CONCLUSIONS: These data demonstrate that iothalamate can replace inulin to measure Jv in microperfusion and free-flow micropuncture experiments. Since iothalamate analysis by CE technique is a fast, easy and highly reproducible technique, it may become the gold standard method for the detection of fluid reabsorption in microperfused nephron segments.

High-performance liquid chromatographic determination of p-aminohippuric acid and iothalamate in human serum and urine: comparison of two sample preparation methods.

A high-performance liquid chromatography method applied to determine p-aminohippuric acid (PAH) and iothalamate (IOT) in serum and urine samples of patients was evaluated according to recovery, reproducibility and linearity utilizing narrow-bore columns. The mobile phase consisted of 0.15 M sodium dihydrogenphosphate with 1.2 mM tetrabutylammonium sulphate, the pH was adjusted to pH 4.6, acetonitrile was added to a final ratio of 95:5 (v/v), the flow-rate was set at 0.3 ml/min. The separation was achieved on a ODS Hypersil column (200 x 2.1 mm I.D.). The UV detector was set at 254 nm. PAH and IOT are used for evaluation of kidney function [effective renal plasma flow (ERPF) and glomerular filtration rate (GFR)]). Under the described chromatographic conditions two sample preparation techniques, ultrafiltration and acetonitrile precipitation were compared. The results demonstrate the accuracy of both methods in evaluation of ERPF and GFR. Due to its cost-effectiveness we recommend the acetonitrile precipitation method in clinical routine.

Determination of iothalamate in rat urine, plasma, and tubular fluid by capillary electrophoresis.

A method for the quantitative determination of iothalamate (IOT) in rat urine, plasma and tubular fluid by capillary zone electrophoresis (CE) has been developed and validated. Samples of urine and tubular fluids were diluted with water and samples of plasma were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 254 nm. The samples were loaded into uncoated fused-silica capillary (40 cmx50 microm) by pressure injection. A borate buffer [20 mM, pH 12 (pH adjusted with 1.0 M NaOH)] was used as the electrophoretic buffer. The typical analytical conditions were: voltage, 22 kV; injection, 9 psixs; capillary and carousel temperatures were 20 degrees C and 18 degrees C respectively. The linear relationship was observed between time-corrected peak area of IOT in water and urine or the corrected peak area ratio of IOT to PABA in plasma and the nominal concentration of IOT with correlation coefficient greater than 0.999. The intra- and inter-day coefficients of variation (CV) were less than 8%. The concentration of IOT in plasma, urine and tubular fluid determined by CE can be used for estimation of whole kidney and single nephron clearances.