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.


METHODS
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.


CONCLUSIONS
This method provides a straightforward means of analyzing multiple anions in small volume biological samples.
Faria
Nuno J R
NJ
School of Biological and Chemical Sciences, Birkbeck College, London, UK. n.faria@bbk.ac.uk
Dobbie
Hamish
H
Slater
Jonathan M
JM
Shirley
David G
DG
Stocking
Christopher J
CJ
Unwin
Robert J
RJ
D000818:Animals; D000838:Anions / Q000032:analysis*; D001639:Bicarbonates / Q000032:analysis; D001826:Body Fluids / Q000737:chemistry; D002712:Chlorides / Q000032:analysis; D019075:Electrophoresis, Capillary; D007483:Iothalamic Acid / Q000032:analysis; D007684:Kidney Tubules / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D010710:Phosphates / Q000032:analysis; D051381:Rats; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet
D000838:Anions; D001639:Bicarbonates; D002712:Chlorides; D010710:Phosphates; D007483:Iothalamic Acid