Determination of Lactate with Liquid Chromatography/Electrochemistry Coupled with a Lactate Oxidase IMER

Liu Yang*, An LCEC (liquid chromatography/electrochemistry) method was Gary Overdorf, and Pete Kissinger developed for the determination of lactate. The method was based on the Bioanalytical Systems combination of anion exchange separation, lactate oxidase IMER West Lafayette, IN 47906-1382 (immobilized enzyme reactor) oxidation of lactate to H2O2, and Phone: (765) 463-4527 electrochemical detection of H2O2 with a peroxidase modified electrode. Fax: (765) 497-1102 E-mail: This method can separate lactate very well from oxidizable anions such [email protected] as ascorbate. The detection limit of the method is 10 µM of lactate with * Present Address: 5 µL injection, and the linear range is 10 µM to 1.5 mM. Lactate in rat Eli Lilly subcutaneous microdialysate was determined with this method. Tippecanoe Labs Lafayette, IN

There have been several methods lactate has to be conducted in the describe the development of a sim- published for the determination of low wavelength UV region. This ple and practical LCEC method for lactate using liquid chromatogra- makes serious background interfer- the determination of lactate. phy. One method (1) involves re- ences likely. There is also a reverse- verse-phase separation with a phos- phase separation using a mixture of Experimental phate buffer at pH 3 as the mobile phosphate and acetonitrile as mo- phase. Following the separation, the bile phase. For this separation, Materials lactate is converted to hydrogen there are two detection methods: Lactate oxidase was purchased peroxide by a lactate oxidase IMER UV-VIS spectrophotometry (3) and from Genzyme (Cambridge, MA). and then detected amperometri- fluorimetry (4). For both detection L-(+)-lactic acid, sodium salt was cally. The optimal pH for the en- methods, the lactate has to be deri- purchased from Calbiochem Cor- zyme lactate oxidase is around 7, vatized and therefore the methods poration. All other chemicals were and at pH 3 the enzyme is basically are not convenient. Ion chromatog- analytical reagent grade from not functioning. Therefore, for this raphy has also been used for the Sigma or Aldrich. Artificial CSF system, a second pump and an on- separation of lactate. The two pub- solution was prepared as follows: line mixer have to be used to in- lished methods use conductimetric 126 mM NaCl, 27.5 mM NaHCO3, crease the pH of the mobile phase (5) and acoustic wave (6) detection. 2.4 mM KCl, 0.5 mM KH2PO4, 1.1 after the separation column to keep The conductimetric method re- mM CaCl2, 0.85 mM MgCl2, 0.5 the enzyme in the IMER active. quires several ion-exchange steps to mM Na2SO4, pH 7.5. All standard This makes the system rather com- reduce the background, resulting in lactate solutions were prepared plicated. Another method (2) also a very complicated system. Be- with the artificial CSF solution. involving reverse-phase separation sides, the chromatogram usually in- with low pH phosphate buffer uses volves many anionic peaks that Microdialysis UV detection instead of the electro- make data analysis difficult. The The rat was anesthetized with chemical detection coupled with a acoustic wave method, on the other an intraperitoneal injection of 1 lactate oxidase IMER. Lactate does hand, is limited by its lack of com- mL/kg of KX (10 mL ketamine not absorb in the usual UV-VIS re- mercial availability and difficult (100 mg/mL) + 1 mL xylazine (100 gion, and the direct detection of data interpretation. In this paper we mg/mL)). The microdialysis probe

15 Current Separations 16:1 (1997) F1 (DL-5, MF-7051, BAS) was placed in the “BAS Beekeeper” contain- Chromatogram of a 5 in an introducer cannula (MF-7021, ment and swivel system (MD-1575, µL injection of a 0.1 300 BAS). The animal’s fur was clipped BAS). All of the experimental pro- mM lactate standard with a 10 cm anion- from the insertion site on the back tocols were approved by the BAS exchange column at the base of the neck. Small inci- Animal Care and Use Committee. and 50 mM 250 Na2HPO4, pH 8.0 as sions were made at both sites. The the mobile phase. Chromatography ) 200 cannula was inserted at the neck in- Flow rate = 1 The LCEC determination of mL/min. Room tem- nA cision and advanced under the skin

( perature. i for about 7 cm. The cannula was lactate was performed with a BAS 150 then pulled back out, leaving the fi- 480 liquid chromatography system ber portion of the probe under the and a peroxidase enzyme electrode 100 skin. The probe was sutured in (MF-2095, BAS) poised at +100 place and perfused with artificial mV vs. Ag/AgCl. An experimental 50 CSF solution at 5 µL/min with a polymeric anion exchange column × BAS Bee syringe drive (MD-1001). (10 0.2 cm, 10 µm) packed at 05During microdialysis sampling, the BAS was used for the separation. min awake unrestrained rat was housed The mobile phase was either 50 mM Na2HPO4, pH 8.0 or 50 mM F2 LiAc, pH 8.25. A lactate oxidase Overlapping chroma- A B IMER made by BAS was used be- tograms of 0.1 mM, 5 tween the separation column and µL lactate (upper) and ascorbate 400 400 the peroxidase electrode detector (lower) A) At the for the oxidation of lactate to hy- same condition as in F1; B) At the same drogen peroxide. In the reverse- condition as in F1 ex- 300 300 phase separation test, a BAS Phase cept that the flow rate was 0.5 mL/min; II ODS-3 column (MF-6215) was C) At the same condi- i (nA) used. The mobile phase was 20 mM tion as in B except 200 200 that the temperature NaH2PO4 and 0.05% (v/v) di- was 35°C; D) With methylhexylamine, pH 5.5 at a flow 20 cm anion-ex- change column and 100 100 rate of 0.5 mL/min. All other condi- at a flow rate of 0.4 tions were the same as with the ion- mL/min. The arrow in- dicates the negative exchange separation. ascorbate peak Unless stated otherwise, all ex- 0 5 0 5 ↑↑periments were carried out at room temperature. C D Results and Discussion 400 150 Because there were two en- 300 zymes (lactate oxidase in the IMER ) and horseradish peroxidase at the

nA 100

(

i electrode) involved in our LCEC 200 system for lactate determination, a phosphate buffer at pH 8, which is 50 generally suitable for enzymes, was 100 first used as the mobile phase. F1 shows a typical chromatogram of a ↑ standard lactate/CSF solution when 0 ↑ 55100 the phosphate buffer was used with min min the ion-exchange column, the lac- tate oxidase IMER, and the peroxi- dase electrode. The graph is clean and simple, however, the separation efficiency of the system was poor. The retention of the lactate was not long enough. With this system, the lactate could not be separated from ascorbate as shown in F2A, where

Current Separations 16:1 (1997) 16 F3 the chromatograms of a lactate so- ficiency. Finally, two separation Chromatogram of 250 lution and an ascorbate solution columns were used instead of one standard 0.1 mM lac- tate and ascorbate solu- taken separately were overlapped. to increase the column length. Due tions, 5 µL, with a 10 200 In this graph, the large positive to the increased back pressure with cm anion-exchange col- umn and 50 mM LiAc, peak is from the 0.1 mM lactate so- the longer column, a flow rate of pH 8.3 as the mobile lution; the negative peak is from the 0.4 mL/min was used in this test. phase. Flow rate = 0.5 150 mL/min. Room tempera- 0.1 mM ascorbate solution. The as- The retention for lactate was much ture. i (nA) corbate peak is actually on the longer with the longer column. 100 shoulder of the lactate peak. The However, the peak was very broad small positive peak from the ascor- and not well shaped. Overall, with 50 bate solution is from the blank so- the phosphate buffer as the mobile lution and will be discussed below. phase, the 10 cm ion-exchange col- 0 ↑ For ion-exchange separation, umn with a flow rate at 0.5 mL/min 010there are several strategies for im- and at room temperature worked proving the separation efficiency, best. min including a lower flow rate, a For further improvement of the higher temperature, and a longer separation, a 50 mM LiAc solution F4 column. Therefore, first, the flow of pH 8.3 was tested as the mobile Chromatogram of a standard lactate solu- 120 rate was reduced from 1 mL/min to phase. The reason for this change of tion of 0.1 mM, 5 µL, 0.5 mL/min with all other condi- mobile phase is that Li+ is a much with an ODS-3 column + and 20 mM NaH2PO4, 100 tions being the same as for F2A.As weaker cation than Na and thus 0.05% (v/v) dimethyl- F2B hexylamine, pH 5.5 as shown in , a lower flow rate has less interaction with lactate, and the mobile phase. Flow increased the retention time and im- acetate is weaker than phosphate ) 80 rate = 0.5 mL/min.

Room temperature. nA proved the separation of lactate and and therefore has less competition

(

i 60 ascorbate. Then the temperature with lactate. Both of them may con- was increased to 35°C by using a tribute to stronger retention of the 40 temperature controller and a heater lactate on the column. F3 shows the for the columns. Since the enzyme chromatograms of lactate and as- 20 in the IMER could not tolerate high corbate obtained at the same condi- temperatures, the temperature in- tion of F2B except that the sodium crease cannot be very dramatic. As phosphate buffer was replaced with 0105 shown in F2C, the increase in tem- the lithium acetate buffer as the mo- min perature did not make any signifi- bile phase. As can be seen in F3, cant difference in the separation ef- with LiAc buffer, the retention of F5 lactate is much longer while the peak is still in very good shape, and Peak current as a func- 6.0E+06 tion of standard lactate the lactate and ascorbate are very solutions. 5 µL sample injected; 10 cm anion- well separated. exchange column; 50 5.0E+06 Besides ion-exchange separa- mM LiAc, pH 8.3 mo- bile phase. Flow rate = tion, a reverse-phase column with 0.5 mL/min. Room tem- an ion-pairing agent in the mobile perature. 4.0E+06 phase was also tested for the lactate / unit determination. Reverse-phase sepa- rations usually have a higher sepa- 3.0E+06 ration efficiency than ion-exchange separations, and by using an ion- 2.0E+06 paring agent, low pH can be Peak Current avoided for the separation. A BAS ODS-3 column with a phosphate 1.0E+06 buffer with dimethylhexylamine as mobile phase was tested. As shown F4 0.0E+00 in , however, the lactate peak has 00.2 0.40.6 0.8 1 1.2 1.4 1.6 1.8 2 very serious tailing under these conditions. The result from the re- Lactate Concentrate / mM verse-phase separation system is simply not as good as that from the

17 Current Separations 16:1 (1997) F6 tate. One small problem with this Conclusion Chromatogram of 5 method for the lactate assay was µL rat subcutaneous The combination of an anion- microdialysate, with that with the injection of almost any 10 cm anion-ex- 800 blank solution, there was always a exchange column with LiAc buffer change column and as mobile phase, a lactate oxidase 50 mM LiAc, pH 8.3 small peak occurring at exactly the as the mobile phase. same position as the lactate. It is IMER, and a peroxidase electrode Flow rate = 0.5 600 ) is a practical configuration for the mL/min. Room tem- not clear whether this peak is due to perature. nA determination of lactate in biologi- ( residual lactate in the injector or

i 400 syringe. This phenomenon recurred cal samples. even with thorough cleaning of the References injector and syringe. This peak 200 from blank solutions limited the de- 1. T. Yao, N. Kobayashi, and T. Wasa, tection limit for lactate to 10 µM. Electroanalysis, 3 (1991) 495-497. Considering the relatively high lac- 2. J. A. Owens and J. S. Robinson, J. 05 tate concentration in most biologi- Chromatogr., 307 (1984) 380-386. cal samples, however, this method 3. B. Bleiberg, J. J. Steinberb, S. D. min Katz, J. Wexler, and T. LeJemtel, is still practical. The upper limit of J. Chromatgr., 568 (1991) 301-308. ion-exchange separation system, al- the dynamic range of the detection 4. S. Ohmori, Y. Nose, H. Ogawa, K. though it can be used. is about 1.5 mM, as shown in F5. Tsuyama, T. Hirota, H. Goto, Y. Based on the above results, the Yano, Y. Kondoh, K. Nakata, and F6 shows a typical chromatogram S. Tsuboi, J. Chromagr., 566 use of an ion-exchange column when this method was used to de- (1991) 1-8. with LiAc buffer as mobile phase, a termine lactate in rat subcutaneous 5. W. Rich, E. Johnson, L. Lois, P. lactate oxidase IMER, and a per- Kabra, B. Stafford, and L. Marton, microdialysate. The lactate deter- Clin. Chem., 26 (1980) 1492-1498. oxidase electrode offered the most mined in the microdialysis samples 6. P. Chen, L. Nie, S. Yao, J. Chro- satisfactory determination of lac- varied from 0.8 to 1.4 mM. magr. B., 673 (1995) 153-158.

Current Separations 16:1 (1997) 18