Simultaneous determination of lactate and pyruvate in human sweat using reversed-phase high-performance liquid chromatography: a noninvasive approach Simona Biagi a , Silvia Ghimenti b , Massimo Onor c and Emilia Bramanti c * ABSTRACT: The simultaneous determination of lactate and pyruvate in sweat has been performed using reversed phase high- performance liquid chromatography (RP-HPLC) with UV detection at 220 nm. The calibration curves were linear in the inves- tigated range 0.3 - 350 mM of lactate, 0.003- 1 mM of pyruvate. The sensitivity was good with a limit of detection of 0.03 mM for lactate and 0.001 mM for pyruvate. Recoveries evaluated for the entire procedure were 102  0.1 and 96  0.1for lactate and pyruvate, respectively. The method was successfully applied to analysis of sweat in 8 athletes at rest (pilocarpine sweating) and during physical exercise. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: sweat analysis; reversed-phase chromatography; lactate; pyruvate; lactate/pyruvate ratio Introduction Pyruvic and lactic acid are two products of glycolysis. The lactate/ pyruvate molar ratio (L/P) in blood is a reliable marker of cell anaer- obic metabolism that may occur in inborn errors of the mitochon- drial respiratory chain (Debray et al., 2007), cardiovascular diseases (ischemia, hypoxemia, anemia; Neill et al., 1969) and other diseases (Ewaschuk et al., 2002). Lactate monitors are also used in diabetes control (Talasniemi et al., 2008) and rehabilitation (Faude et al., 2009). Oxygen deficits (tissue hypoxia) are the most common and often refractory causes of lactic acidosis, including pulmonary problems (low PO 2 ), circulatory problems (poor delivery of O 2 ) and hemoglobin problems (low O 2 -carrying capacity, for various reasons; Luft, 2001). In muscle, during exertion, pyruvate derived from glycogen is reduced to lactate, which is reoxidized and partially reconverted into glycogen during rest. The concentration of blood lactate is usually 1–2 mmol/L at rest, but can rise to over 20 mmol/L during intense exertion, because of the switch of muscle cells to anaero- bic metabolism. However, once a certain level of lactate concen- tration is reached, exhaustion occurs and there is a rapid decline in exercise capacity (Graham et al., 1987). Thus, in sport medicine it is used to monitor the maximum performance level of athletes (Noy et al., 1978; Sahlin et al., 1987; Sahlin et al., 1992; Mitsubayashi et al., 1994). The determination of lactate and pyruvate is usually per- formed in plasma or urine and based on the enzymatic reaction between lactate and co-factor NAD + (Ewaschuk et al., 2002), which is very fast but not specific, or by gas chromatography (Inoue et al., 2006; Paik et al., 2008), capillary electrophoresis (Tan et al., 2005) or liquid chromatography with mass spectromet- ric detection (LC-MS; Chuang et al., 2009; Franco et al., 2009), high- performance liquid chromatography (HPLC; Okubo et al., 2000; Pailla et al., 2000; Ewaschuk et al., 2002; Cevasco et al., 2011). LC-MS techniques are reliable and fast approaches to lactate analysis in plasma or blood (McKinnon et al., 2006, 2008). However, the cost of instrumentation is typically high and measurements require well-trained personnel. Furthermore, the sampling of blood has to be performed by qualified medical staff with consid- eration for rigid hygienic regulations. The measurement of metabolites in media other than blood is becoming increasingly significant because of major demands for noninvasive analysis. Such measurements are particularly impor- tant to avoid physical and mental strain and infection risk for patients who have to control daily parameters, for people in whom collecting blood is a problem (hemophiliacs, neonates, elderly people), and for athletes to estimate their physical and biochemical condition and to evaluate their training regime. The use of sweat as an analytical sample dates back almost 30 years. The analysis of metabolites present in sweat is safe and simple. The analysis of sweat for electrolyte concentrations still remains the laboratory ‘ gold standard’ for the diagnosis of cystic fibrosis (Tocci and McKey, 1976). * Correspondence to: Emilia Bramanti, National Research Council of Italy, C.N. R., Istituto per i Processi Chimico-Fisici-IPCF- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy. E-mail: bramanti@pi.iccom.cnr.it a National Research Council of Italy, C.N.R., Istituto per i Processi Chimico- Fisici-IPCF- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy b Dipartimento di Chimica e Chimica Industriale, Via Risorgimento 35, 56127 Pisa, Italy c National Research Council of Italy, CNR, Istituto di Chimica dei Composti Organo Metallici-ICCOM- UOS Pisa, Area di Ricerca, Via G. Moruzzi 1, 56124 Pisa, Italy Abbreviations used: AT, aerobic training; BB, boxing bag; Cys, cysteine; GSH, reduced glutathione; GSSG, oxidized glutathione; KP, kick-boxing punch/pads; L/P, lactate/pyruvate molar ratio; PBS, phosphate buffer solution. Biomed. Chromatogr. 2012; 26: 1408–1415 Copyright © 2012 John Wiley & Sons, Ltd. Research article Received: 15 December 2011, Accepted: 3 January 2012 Published online in Wiley Online Library: 7 February 2012 (wileyonlinelibrary.com) DOI 10.1002/bmc.2713 1408