Journal of Chromatographic Science 2015;53:1475– 1480
doi:10.1093/chromsci/bmv040 Advance Access publication April 29, 2015
Article
High-Performance Liquid-Chromatographic Analysis of Plasma Iohexol Concentrations†
Harvey A. Schwertner1* and Kyle J. Weld2
1
Clinical Research Division, Wilford Hall Ambulatory Surgical Center, JBSA Lackland, 2200 Bergquist Drive, San Antonio, TX 78236, USA,
and 2Department of Nephrology, JBSA Lackland, San Antonio, TX 78236, USA
*Author to whom correspondence should be addressed. Email: [email protected]
†
The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Defense or other
Departments of the United States Government.
Received 2 June 2014; revised 3 March 2015
In this study, a high-performance liquid-chromatographic (HPLC)
method using photodiode array detection and isocratic conditions
was developed for the analysis of plasma iohexol concentrations.
Plasma proteins were precipitated with 1:1 volume of plasma and
acetonitrile –ethanol – water (60:38.4:1.6, v/v/v). Iohexol concentrations in the supernatant phase were analyzed on a Waters Symmetry
C-18 reversed-phase column under isocratic conditions at 245 nm.
The extraction recoveries of iohexol from plasma were >95% and
the plasma iohexol calibration curves were linear (R 2 0.9998)
from 10 to 1500 mg/mL. The within-day coefficients of variation
(CVs) at plasma iohexol concentrations of 100, 375, 750 and
1500 mg/mL were 5.1, 3.5, 1.3 and 2.5%, respectively; the
between-day CVs at 100, 375, 750 and 1500 mg/mL were 8.6, 4.2,
4.0 and 3.7%, respectively. The day-to-day accuracies of the method
at plasma iohexol concentrations of 50, 100, 375, 750 and 1500 mg/mL
were 89.0, 99.4, 108.4, 103.6 and 101.2%, respectively (n 5 5). The
lower limit of plasma iohexol quantitation was 10 mg/mL and no interferences >9 mg/mL were found in over 75 pre-dose porcine plasma
samples. The applicability of the method was demonstrated by determining the glomerular filtration rates of iohexol in the porcine
(Sus scrofa) model.
Introduction
Iohexol (Omnipaque, N/N 0 -bis(2,3-dihydroxypropyl)-5-[N-(2,3dihydroxypropyl)-acetamido]-2,4,6-triiodo-isophthalamide) is
an iodinated non-ionic monomeric contrast agent (Figure 1)
that is highly water soluble (1), does not bind to serum proteins
and is completely eliminated through the glomerulus (2 –4). As a
result, it has been used to determine glomerular filtration rates in
humans (2 –7) and in animals (8, 9). Inulin clearance remains the
“Gold Standard” for the determination of GFR in humans; however, its use is complicated by the lack of commercially available
dosage forms, the need for infusion pumps and multisample collections and the reliability of the analytical methods used for its
analysis (7). Radioactive markers such as ethylenediaminetetraaceticacid (51Cr-EDTA), diethylenetriaminepentaaceticacid
(99mTc-DTPA) and 125I-iothalamate have been used as alternatives to inulin (7); however, their use requires radioactive licensing and compliance with regulations governing the storage, use
and disposal of radioactive material. In addition, the radioactive
markers cannot be used in certain patients including women
who are pregnant (7). Iohexol clearance is an attractive
substitute for determining GFR in that it is considered to be almost as accurate as 51Cr-EDTA clearance and the results obtained
with iohexol agree with those obtained with inulin (4).
Several high-performance liquid-chromatographic (HPLC)
methods (9–14) and two liquid-chromatographic mass-spectrometric (LC–MS-MS) methods (15–16) have been developed for
the analysis of iohexol concentrations in plasma. Most of the
HPLC methods require a protein precipitation step with either
perchloric acid (13, 14), trifluoroacetic acid (11, 12), zinc sulfate
(15, 16) or acetonitrile–ethanol–water (12), direct injection of diluted serum (10) or the use of column switching devices to separate iohexol from the plasma proteins (10). In addition, most of
the HPLC methods require the use of gradient chromatography to
achieve separation (9, 11, 12, 15, 16) or the use of both gradient
chromatography and changes in flow rates (13). LC–MS-MS methods are very sensitive and specific (15, 16); however, triple quadrupole mass spectrometers and other mass spectrometers are not
always available in hospital laboratories. Even though the HPLC
methods are relatively simple, insufficient information on the
methods and a lack of rigorous method validation has often led
to problems in reproducing some of the methods.
The aim of this study was to develop a new HPLC method for
the analysis of plasma iohexol that is simple, accurate and reproducible and that can be used to determine glomerular filtration
rates in both animals and humans. In the study, several chromatographic columns, mobile phases and ultraviolet wavelengths were
evaluated for the analysis of iohexol in plasma samples. In addition,
iohexol-related compound B, iopamidol and iothalamate, were
evaluated as possible internal standards. Information on the validation of the HPLC method and the use of the method to determine
iohexol clearance in the porcine model is also given in this report.
The acetonitrile–ethanol–water precipitating reagent used in this
study results in more complete removal of co-extractable interferences, lower injection volumes and greater sensitivity than most
other HPLC methods using ultraviolet detection (9–10, 12–14).
The mobile phase and column used in this study also permits
the analysis of iohexol under isocratic conditions rather than gradient conditions.
Experimental
Reagents and chemicals
Omnipaque 240 iohexol injection, 517.7 mg/mL iohexol, and
Omnipaque 350 iohexol injection, 755 mg/mL iohexol, were obtained from GE Healthcare, Princeton, NJ, USA. Iohexol-related
Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
negative controls, plasma iohexol calibrators and the iohexol
standards were stored at 4 + 38C.
Figure 1. Chemical structure of iohexol. This figure is available in black and white in
print and in color at JCS online.
compound B USP Reference Standard, 50.0 mg, Cat. No. 34464
and iopamidol USP Reference Standard, 200 mg, Cat. No.
34770, were obtained from USP, Rockville, MD, USA. Conray
iothalamate meglumine injection, 180 mg/mL, was obtained
from Mallinckrodt, St Louis, MO, USA. Acetonitrile (Cat. No.
9017-03) was obtained from JT Baker; ethanol (Cat. No. E7023)
and trifluoroacetic acid (Cat. No. T6508) from Sigma-Aldrich, and
methanol (Cat. No. A452-4) from Fisher Scientific.
The 0.1% trifluoroacetic acid (v/v) used in the mobile phase
was prepared by combining 1.0 mL of trifluoroacetic acid with
999 mL of water purified on a Milli Q water purification system
(Millipore, Bedford, MA, USA). The pH of the 0.1% trifluoroacetic
acid was adjusted to pH 2.2 with 1.0 M sodium hydroxide and
then filtered through a 0.2 mm filter. The 0.1% trifluoroacetic
acid–methanol, 80:20, v/v, mobile phase was prepared by mixing
800 mL of 0.1% trifluoroacetic acid and 200 mL of methanol.
Animal studies
Glomerular filtration rates were determined in a porcine pig
model used to identify markers for predicting irreparable ischemic damage. Iohexol was injected via the venous port or with a
Hickman catheter at an iohexol dose of 0.5 mL/kg. Pre-dose and
2, 3 and 4 h post-dose blood samples were collected with an indwelling catheter into 10 mL lithium –heparin vacutainer tubes
at days 1, 5, 9, 14 and 28 for each of 15 pigs. The plasma samples
were stored at 2708C until analyzed. The protocol for the study
was approved by our Institutional Animal Care and Use
Committee (IACUC).
Preparation of iohexol standards and plasma calibrators
Pig plasma was used to prepare the negative plasma control and
the plasma iohexol calibrators at 10, 25, 50, 100 and 375, 750 and
1500 mg/mL. A plasma stock solution containing iohexol at a
concentration of 5177 mg/mL was prepared by diluting 1 mL of
Omnipaque (517 mg/mL) with 99 mL of the negative pooled pig
plasma. To prepare the 1500 mg/mL plasma iohexol calibrator,
29 mL of the 5177 mg/mL plasma iohexol stock solution was
mixed with 71 mL of the pooled pig plasma sample. Dilutions
of the 1500 mg/mL plasma iohexol calibrator were used to prepare the 750, 375, 100, 50, 25 and 10 mg/mL plasma iohexol calibrators. Iohexol standards were prepared in the same fashion,
but were made up in methanol rather than in plasma. The plasma
1476 Schwertner and Weld
Plasma protein precipitation procedure
All steps in the protein precipitation were performed in a walk-in
refrigerator at 4 + 38C. Protein precipitation was performed by adding 400 mL of each plasma calibrator or plasma test sample and
400 mL of the protein precipitating reagent (acetonitrile–ethanol–
water, 60.0:38.4:1.6, v/v/v) to Fisherbrand microcentrifuge tubes
(1.5 mL polypropylene with indented cap, Cat. No. 02-681-2391,
Fischer Scientific, Fair Lawn, NJ, USA). The samples were then
mixed on a vortex mixer for 10 s and kept at 4 + 38C for 2 h.
The plasma samples were then centrifuged in an Eppendorf
Minispin Plus centrifuge (Model 5453, Hamburg, Germany) at
14,100 RCF for 10 min to precipitate the proteins. The supernatant
phase of each sample was then transferred with a Pasteur pipet to
separate plastic microcentrifuge tubes and centrifuged again at
14,100 RCF for 10 min. The clear supernatant phases were then
transferred to 700 mL glass HPLC injection vials, capped and analyzed or stored at 4 + 38C until analyzed by HPLC.
HPLC chromatographic analysis
The iohexol standards, plasma iohexol calibrators and plasma test
samples were analyzed on a Waters Chromatographic System consisting of a Waters 2695 Separations Module, 2996 Photodiode
Array Detector (PDA), and a Millennium 32 Chromatography
Manager (Waters, Milford, MA, USA). Chromatographic analysis
was performed on a 4.6 250 mm Waters Symmetry C-18
reversed-phase column (Cat. No. WATO 54215) with 0.1%
trifluoroacetic acid (pH 2.2)–methanol (80:20, v/v) as the mobile
phase. The HPLC operating parameters were as follows: injection
volume, 5.0 mL; column flow rate, 0.3 mL/min; chromatographic
run time, 16.0 min; PDA spectra recording, 245 nm. The iohexol
peak identifications were based on the retention times of the
iohexol standards and further confirmed by comparing their photodiode array spectra to those of the iohexol standards.
Results
In this study, various types and sizes of C-8 and C-18 reversedphase columns were evaluated; however, the 4.6 250 mm
C-18 column was found to result in sharper peaks, greater sensitivities and better resolution of iohexol from co-extractable interferences than comparable C-8 columns. HPLC chromatograms
achieved with this column for a plasma iohexol calibrator, a predose plasma sample and a post-dose plasma sample are shown in
Figure 2. As shown, there are few peaks other than iohexol even in
the pre-dose plasma samples. With this column and mobile phase,
iohexol was separated into its two isomeric forms. Even though
both peaks could have been combined and used to increase the
sensitivity levels, the larger iohexol isomer was used to quantitate
plasma iohexol concentrations. The smaller peak typically had a
peak area about 20% that of the larger peak.
The standard curves for plasma iohexol were linear from 10.0 to
1500.0 mg/mL. For quantitation of glomerular filtration rates, standard curves were prepared with plasma iohexol concentrations at
50, 100, 375, 750 and 1500 mg/mL (see Supplementary Material).
Figure 2. HPLC chromatograms of plasma iohexol calibrator, negative plasma calibrator and plasma sample analyzed to determine glomerular filtration rate. (A) Plasma iohexol
calibrator, 375 mg/mL. (B) Pre-dose plasma sample. (C) Three-hour post-dose baseline plasma sample. Chromatographic analyses in (A – C) were performed under isocratic
conditions on a 4.6 250 mm Waters Symmetry C-18 reversed-phase column (Cat. No. WATO 54215) with 0.1% trifluoroacetic acid ( pH 2.2)-methanol (80:20, v/v) as the
mobile phase. The HPLC operating parameters were as follows: injection volume, 5.0 mL; column flow rate, 0.3 mL/min; chromatographic run time, 16.0 min; PDA spectra
recording, 245 nm.
Five standard curves used for the studies had an R 2 of 0.9998 +
0.0005 (mean + SD, y ¼ 13.761x þ 165.1). Within-day and
between-day imprecision (CVs) were determined by analyzing
multiple spiked plasma samples at iohexol concentrations of
50, 100, 375, 750 and 1500 mg/mL. Single injections were used
for the determination of all CVs, accuracies and test results. The
HPLC Analysis of Plasma Iohexol 1477
within-day CVs for plasma iohexol at 100, 375, 750 and 1500 mg/
mL (n ¼ 5) were 5.1, 3.5, 1.3 and 2.5%, respectively, and the
between-day CVs at 100, 375, 750 and 1500 mg/mL were 8.6, 4.2,
4.0 and 3.7%, respectively (n ¼ 7). Accuracies were determined by
comparing the actual iohexol concentration (mg/mL) to the determined concentration over 5 days. The day-to-day accuracies of the
method at 50, 100, 375, 750 and 1500 mg/mL were 89.0, 99.4,
108.4, 103.6 and 101.2%, respectively (n ¼ 5).
The specificity of the method was determined by analyzing 75
pre-dose plasma samples. These samples were obtained from 15
different pigs. Of the 75 pre-dose plasma samples analyzed,
only 2 plasma samples had measureable amounts of iohexol
(1.0 – 9.0 mg/mL). When the two plasma samples were reanalyzed, they did not contain any measureable amounts of iohexol
(6.0 mg/mL). Likewise, all of the other pre-dose plasma samples did not contain any measureable amounts of iohexol
(6.0 mg/mL). Likewise, extraneous peaks that might interfere
with iohexol analysis were not found in over 200 post-dose samples or in over 100 plasma iohexol calibrators. Photodiode array
scans from 200 to 400 nm also indicated that the two iohexol isomers had ultraviolet spectra like those of the iohexol standards
and were free of other co-eluting substances.
The sensitivity of the method was established by determining the
lower limit of quantitation and the limit of detection. The lower limit
of quantitation (LLOQ) was determined as the minimum plasma
iohexol concentration that can be quantitatively determined with
a peak height to base line ratio of at least 10:1. The limit of detection
as established as a peak height to base line ratio of at least 3:1. As a
result of these analyses, the limit of detection and the lower limit of
quantitation were found to be 6.0 and 10.0 mg/mL, respectively.
No efforts were made to increase the sensitivity by injecting larger
volumes of the supernatant phase, altering the ratio of the plasma to
precipitating agent volumes, combining the iohexol isomers or further purifying the supernatant phases.
As determined by UV scans, the maximum ultraviolet absorbance of iohexol was at 245 nm (Figure 3). There were no differences in UV absorptivity when dissolved in 0.1% TFA ( pH 2.2)–
methanol, 80:20, v/v, or when dissolved in water (Figure 3). In
this study, plasma iohexol was initially measured at both 245
and 255 nm and the resulting specificities and sensitivities at
the two wavelengths were found to be much the same. As a result, the method validations and test sample analyses were
Figure 3. UV absorbance of iohexol (0.01 mg/mL) in 0.1% TFA ( pH 2.2) – methanol
(80:20, v/v) and in water. UV scans of iohexol performed in the HPLC mobile phase
(0.1% TFA, pH 2.2)– methanol (80:20, v/v) (see Figure 2) and in water. This figure is
available in black and white in print and in color at JCS online.
1478 Schwertner and Weld
performed at 245 nm. The chromatographic column and mobile
phase used in this procedure resulted in retention times that
were very similar from day to day. Plasma iohexol calibrators
and plasma iohexol calibrator protein-free precipitates were
found to be stable for at least 5 days at 4 + 38C.
A number of closely related compounds were evaluated as possible internal standards and included iohexol-related compound
B, iopamidol and iothalamate. Iohexol-related compound B, iopamidol and iothalamate were all soluble in water; however, only
iopamidol was soluble in the protein precipitation reagent. It
took 3 days to dissolve the iopamidol crystals in the protein
precipitating reagent at room temperature and to produce a
clear solution. All three internal standards exhibited excellent
peak shape and sensitivity when dissolved in water, but only
iopamidol had a narrow chromatographic peak shape and good
sensitivity when dissolved in the protein precipitating reagent.
The protein precipitating reagent containing iopamidol was
added to plasma calibrators containing 0, 125, 250, 500, 1000
and 1500 mg/mL of iohexol and then analyzed by HPLC. The
plasma iohexol standard curve obtained with the iopamidol
containing protein precipitating reagent had an R 2 0.9954.
Accuracies of iohexol obtained with iopamidol were not quite
as good as those obtained without an internal standard. As a result, analyses of iohexol in the test samples were performed without the use of iopamidol as an internal standard and further
method validations with iopamidol were not performed.
Plasma iohexol concentrations at 2, 3 and 4 h were determined
on 75 pigs with varying degrees of ischemic renal damage.
Typical plasma iohexol concentrations for four normal pigs are
shown in Figure 4.
Discussion
The HPLC method for analyzing plasma iohexol concentrations is
simple and, as shown here and elsewhere (17), can be used to
Figure 4. Plasma iohexol clearance. This figure is available in black and white in print
and in color at JCS online.
determine the glomerular filtration rates. The method has a relatively low within-day and between-day CVs and there were no
co-extractable substances found in any of the pre-dose pig plasma
samples at concentrations 9.0 mg/mL. The lower limit of quantitation was found to be 10.0 mg/mL. The LLOQ was comparable
with the 10.0 mg/mL reported by Farthing et al. (11) and Soman
et al. (13) and better than the 25.0 mg/mL limit found by Shihabi
et al. (10). The method is sufficiently sensitive to determine
iohexol clearance in humans. Plasma iohexol concentrations measured between 0.25 and 7.0 h after an intravenous administration
of a standard dose of iohexol (10 mL, 647 mg of iohexol/mL) was
found to be 272.5 + 133.1 mg/mL (concentration range 33.9 –
674.0 mg/mL) (13). The minimum plasma iohexol concentration
measured at 7 h post-dose was 123.5 + 61.1 mg/mL (concentration range 33.9 – 225.4 mg/mL). These iohexol concentrations
are well within the sensitivity limits of our method as well as within the linear limits of our standard curves.
In the current study, acetonitrile – ethanol – water (60.0:38.4:
1.6, v/v/v) was used as the protein precipitating reagent whereas
others used trifluoroacetic acid (11, 12), perchloric acid (13, 14)
or zinc sulfate (15, 16). Acetonitrile – ethanol – water has been
used in a previous study (12); however, only limited method validations were performed in that study. Trifluoroacetic acid has
been used as a protein precipitating reagent in two studies; however, the trifluoroacetic acid concentrations varied considerably
in the two studies (11, 12). One of the studies, for example, used
0.1% trifluoracetic acid (11), whereas the other study used 20%
trifluoroacetic acid (12). Several other methods did not use a protein precipitating reagent but rather injected diluted plasma samples directly on to the column (8, 10).
Iopamidol worked effectively as an internal standard; however,
its accuracies were slightly lower than the accuracies performed
without an internal standard. While iohexol-related compound B
and iothalamate were not soluble in our protein precipitating reagent, they and other similar compounds could possibly be used
with modifications to our protein precipitating reagent, with
other protein precipitating reagents (trifluoroacetic acid, perchloric acid, etc.), or with solid-phase extraction procedures.
The HPLC method described here has advantages over other
HPLC methods in that it is an isocratic method, whereas other
methods utilized gradients (9, 11, 12, 15, 16) or a combination
of gradients and changes in flow rates to achieve separation of
iohexol from co-eluting material (13). Although the previously
published HPLC methods resulted in complete separation of
iohexol from co-eluting substance, the HPLC chromatograms
obtained with those methods (9 –13) contained more chromatographic peaks than found with our method. The iohexol concentrations in the current work’s standard curve ranged from 10.0 to
1500 mg/mL, whereas those described in other methods ranged
from 10.0 to 50.0 mg/mL (11) and 10.0 to 750.0 mg/mL (13). The
reported LC – MS-MS methods; however, had linear ranges to
1500.0 mg/mL (15, 16). Our method also differed from that of
other methods in that our injection volume was 5.0 mL.
Farthing et al. (11) used an injection volume of 10 mL; however,
Soman et al. (13) had to use 90.0 mL injection volumes to achieve
sufficient sensitivity.
The HPLC method described here has been used for determining glomerular filtration rates in our animal studies (17) and
should be applicable to determining glomerular filtration rates
in humans. The precipitation method is simple and results in
few co-extractable components at 245 or at 255 nm compared
with other published methods (13). Unlike other studies (13),
differences in baselines at 245 and 255 nm were not found in
this study. It could be that the acetonitrile –ethanol –water precipitating reagent is more effective in precipitating proteins than
is perchloric acid (13, 14).
Conclusion
The HPLC method described here is accurate, reproducible
and easy to perform and has sufficient sensitivity and specificity
for the determination of glomerular filtration rates in animals
and in humans. The method uses protein precipitation with
acetonitrile – ethanol – water (60.0:38.4:1.6, v/v/v) followed by
HPLC analysis on a C18 column using isocratic conditions.
Iohexol-related compound B, iopamidol and iothalamate were
evaluated as possible internal standards. Analyses performed
with iopamidol were found to be less accurate than analyses performed without an internal standard. This could be due to the
low solubility of iopamidol in the precipitating reagent. The analytical method was used to determine iohexol clearance in pigs
with and without renal ischemic injury.
Supplementary material
Supplementary materials are available at Journal of
Chromatographic Science (http://chromsci.oxfordjournals.org).
References
1. Foster, S.J., Sovak, M.; Isomerism in iohexol and ioxilan. Analysis
and implications; Investigative Radiology, (1988); (Suppl. 1):
S106–S109.
2. Stake, G., Monclair, T.; A single plasma sample method for estimation
of the glomerular filtration rate in infants and children using iohexol.
1: Establishment of a body weight-related formula for the distribution
volume of iohexol; Scandinavian Journal of Clinical and Laboratory
Investigation, (1991); 51: 335–342.
3. Stake, G., Monn, E., Rootwelt, K., Monclair, T.; A single plasma sample
method for estimation of the glomerular filtration rate in infants and
children using iohexol. II: Establishment of the optimal plasma sampling time and a comparison with the 99T cm-DTPA method;
Scandinavian Journal of Clinical and Laboratory Investigation,
(1991); 51: 343– 348.
4. Ericksson, C.G., Kallner, A.; Glomerular filtration rate: a comparison
between Cr-EDTA clearance and a single sample technique with
a non-ionic contrast agent; Clinical Biochemistry, (1991); 24:
261–264.
5. Brown, S.C., O’Reilly, P.H.; Iohexol clearance for the determination of
glomerular filtration rate in clinical practice: evidence for a new gold
standard; The Journal of Urology, (1991); 146: 675– 679.
6. Krutzen, E., Olofsson, P., Back, S.E., Nilsson-Ehle, P.; Glomerular filtration rate in pregnancy: a study in normal subjects and in patients
with hypertension, preeclampsia and diabetes; Scandinavian
Journal of Clinical and Laboratory Investigation, (1992); 52:
387 – 392.
7. Frennby, B., Sterner, G.; Contrast media as markers of GFR; European
Radiology, (2002); 12: 475– 484.
8. Finco, D.R., Braselton, W.E., Cooper, T.A.; Relationship between
plasma iohexol clearance and urinary exogenous creatinine clearance in dogs; Journal of Veterinary Internal Medicine, (2001); 15:
368–373.
HPLC Analysis of Plasma Iohexol 1479
9. Meucci, V., Gasperini, A., Soldani, G., Guidi, G., Giorgi, M.; A new HPLC
method to determine glomerular filtration rate and effective renal
plasma flow in conscious dogs by single intravenous administration
of iohexol and p-aminohippuric acid; Journal of Chromatographic
Science, (2004); 42: 107–111.
10. Shihabi, Z.K., Thompson, E.N., Constantinescu, M.S.; Iohexol determination by direct injection of serum on the HPLC column; Journal of
Liquid Chromatography, (1993); 16: 1289–1296.
11. Farthing, D., Sica, D.A., Fakhry, I., Larus, T., Ghosh, S., Farthing, C.,
et al.; 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; Journal of Chromatography B,
(2005); 826: 267–272.
12. Jacobsen, P.B.; High performance liquid chromatography with
multiwavelength detection: A technique for identification of
iodinated x-ray contrast agents in human body fluids and brain
tissue; American Journal Neuroradiology, (1992); 13: 1521– 1525.
1480 Schwertner and Weld
13. Soman, R.S., Zahir, H., Akhlaghi, F.; Development and validation of an
HPLC-UV method for determination of iohexol in human plasma;
Journal of Chromatography B, (2005); 816: 339–343.
14. Krutzen, E., Back, S.E., Nilsson-Ehle, P.; Determination of glomerular
filtration rate using iohexol clearance and capillary sampling;
Scandinavian Journal of Clinical and Laboratory Investigation,
(1990); 50: 279– 283.
15. Lee, S-Y., Chun, M-R., Kim, D-J., Kim, J.W.; Determination of iohexol
clearance by high-performance liquid chromatography-tandem mass
spectrometry (HPLC-MS/MS); Journal of Chromatography B,
(2006); 839: 124– 129.
16. Annesley, T.M., Clayton, L.T.; Ultraperformance liquid chromatography-tandem mass spectrometry for iohexol in human serum;
Clinical Chemistry, (2009); 55: 1196–1202.
17. Weld, K.J., Montiglio, C., Bush, A.C., Dixon, P.S., Schwertner, H.A.,
Hemsley, D.M., et al.; Predicting irreparable renal ischemic injury
using a real-time marker in the porcine model; The Journal of
Urology, (2008); 22: 18–25.