IUBMB Life, 51: 231–239, 2001 c Copyright ° 2001 IUBMB 1521-6543/01 $12.00 + .00

Critical Review

The Relevance of Flow Cytometry for Biochemical Analysis

Jose-Enriqu´ e O’Connor,1 Robert C. Callaghan,2 Marta Escudero,1 Guadalupe Herrera,1 Alicia Mart´õ nez,1 Mar´õ a-do-Ceu´ Monteiro,3 and Hilario Montol´õ u1 1Centro de Citometr´õ a, Departamento de Bioqu´õ mica y Biolog´õ a Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain 2Departamento de Patolog´õ a, Facultad de Medicina, Universidad de Valencia, Valencia, Spain 3Departamento de Bioqu´õ mica, Instituto Superior de Cienciaˆ s da Saude-Norte´ , Gandrˆ a PRD, Portugal

as they  ow rapidly through a sensing area (1, 2). In some sys- Summary tems (cell sorters), individual cells or particles may be physically Flow cytometry (FCM) allows the simultaneous measurement separated according to their properties (Fig. 1). Thus, FCM is of multiple  uorescences and light scatter induced by illumination unique in that multiple biological parameters can be quantiŽ ed of single cells or microscopic particles in suspension, as they  ow simultaneously on a single-particle basis, while up to thousands rapidly through a sensing area. In some systems, individual cells of events per second may be examined. As a result, large and or particles may be sorted according to the properties exhibited. By using appropriate  uorescent markers, FCM is unique in that heterogeneous cell populations are described based on the bio- multiple structural and functional parameters can be quantiŽ ed si- metric properties of their individuals (Table 1). multaneously on a single-particle basis, whereas up to thousands of Because of its historical development (1) and its important biological particles per second may be examined. FCM is increas- clinical implications, the largest body of current applications is ingly used for basic, clinical, biotechnological, and environmental diagnostic/prognostic, based on immunophenotyping and DNA studies of biochemical relevance. In this critical review, we summa- rize the main advantages and limitations of FCM for biochemical content assays (3, 4). However, FCM is now a choice methodol- studies and discuss brie y the most relevant parameters and ana- ogy in basic and applied studies, including cellular and molec- lytical strategies. Graphical examples of the biological information ular biology (5, 6), biotechnology (7), toxicology (8), microbi- provided by multiparametric FCM are presented. Also, this review ology (9), plant physiology (10), and oceanography/limnology contains speciŽ c sections on  ow cytoenzymology, FCM analysis of (11). On the other hand, clinical FCM increasingly implements isolated subcellular organelles, and cell-free FCM. IUBMB Life, 51: 231–239, 2001 biochemical assays to improve sensitivity of abnormal cell iden- tiŽ cation (12) and toprovide functional information about patho- genetic mechanisms involved in disease conditions (13). Keywords Cell function; cell sorting;  ow cytometry;  uorescence;  uorochromes; isolated subcellularelements; multiplexed assay. FCM Analysis of Cell Biochemistry: Parameters and Probes INTRODUCTION Individual cells, bearing multiple markers on their surface, Flow cytometry (FCM) allows the simultaneous measure- contain intracellular compartments with their own metabolic ment of multiple  uorescences and light scatter induced by illu- environment. Functional integrity of membranes is necessary mination of single cells or microscopic particles in suspension, for the regulation of such compartments that, in turn, condition metabolic pathways within them. In many cases, homeostasis of cell compartments requires regulated transport across cell mem- branes. On the other hand, plasma membrane is deeply involved Accepted 24 May 2001. in biochemical responses that mediate cell activation triggered Address correspondence to Dr. Jose-Enriqu´ e O’Connor, Centro de Citometr´õ a, Departamento de Bioqu´õ mica y Biolog´õ a Molecular, Facul- by multiple stimuli. Many of these responses are dependent on tad de Medicina, Avda. Blasco Iba´nez˜ , 17. 46010-Valencia. Fax: +34- speciŽ c receptors and trigger consistent changes in ionic status 963-864186. E-mail: [email protected] and enzyme activities in signal transduction pathways, leading

231 232 O’CONNOR ET AL. . , r r s s e y d o r ) t e o k h a e s n t m r a b s g e g T e m o r t n l n . m e i s m i w a d s t i t a a t y l l t c t n r l i e s i h e p a r a e i c s n e t t m e c ) p l l r r , d m a u w m e e o r n l o b s o p u a m s ( o g o  n i d l o y n t g n t n i i a g e d I n s o a n i e . r h t w h t t s Ž n o g r S p e a e t Ž l n o l i y d a e s e a c n r t i n i l c t d e i t a l - t e o r ) m r a r e l a t s a a a c e m g e n e p p s y l o d o s b e r u b t Ž n c a S o y r r e a e . e s r s h o C ) t l d s y e c l ) s l e a o . h u s l e l g w b s s c k p o n m w n , l a i s t m o e t i s ) o s r e l F t i i i s s d ( s t d l e s g t s s ( s a s y n l o o a D l i l l c o r e a l i t s l a r p c l C u e t y n e Ž y p , d c t c o a o i s l b p ( t S d e v k e a Ž l s i d s C i d e h g i e d t t d s e t F d n M n a n y f c i a i i l s r a r s C r o y a a o e c a F p s s f i n h n v d p n e r a i c p e r e t e e o o a r o t l l r g u f t e  c o t u a s a ( f g s a e r e s r m n o e s s o m s l i r f a f l l r g t r c m e u c o n e i o w x e i f t c e p i t t f l s m r m t s l e t e o e o r y w o m a s h o s s a s t o c t a l r m t y h d s n s a t e r i i g t e e r a c s s o r o t n a i Ž t n y p a i w e w s l p s e p s - i d o a e o c s m u d h  n d h s d a s r u s l a e i r s e e l e r t t c t h f a e a h s t o a a t o d l i c n g u f i m r i e n s n s  g o e a r t a f i l r y n v h o g e e i s i u o h c s l n h e s n k c s p t s u a o e a u n r s r ( h e e y h t e o e r c h b e g n h n c n b t r h T u o o o d s o i r c r o T g . e a t r t e r p e . n g t u h i n d e f s h n t n b f e t e d i a i c o s r i r i u d p s t m n l s t o n e e i s t a c u t e w i e o s s r r c c n i o r t t d k i i t l e e s u l v r c , d e n b n i q a l e s e i n o i i c l d d i d y a s a h e t g a n d y t a g d a l n w a l n t i n m e n a , t u a i e a r e s r n a p a g d s t d a o m r m o h i e a r s e a t u s p a t c e r t l h l D t c l t l o e a c a l e e . r s e b l d e e e p l n c c r m r i c / r i y o a t a n e y n f a e c r i a r o h t o w t h a i t e m s t t t s e p r l r f s i i o s a u d s a t o n r m i e f l t n n s l o s o u u i c l a g t o Ž t d i q e t a y m a y l s e c n t l c t S e d a r d g g n a e C i o e a e c n y w e f s i t s F i s b c o s a l d r . d e o e r  r d o e e n d r t c c p o d l t e o e p o c t e o a a h r t r y a c o t c o c r p o t i s b a s c r a y i p e s t s l d d p p l h r y t h h e n t e t a e p n e g a t s n s i a a h m a , u t l r t r g e e s y i p f g o v f d b s a i n f o t l o i n m r e e n y p a e s o c d n h s e a c c e p t i r n e o i r i t t d m e a c s y t r f i a n i f n f f e s y i e n i e t s l l l o m i l l d d c u Ž p e s e a e s n e p h s y c u e p m e d l c a t r m y a p ) r e a t S o i r s o e d s n , r u v c p e d e a e n .  n y d t e s l d o i 1 r a c o a a e i e l i t r l o c c u l p e i s a e d l p a d t l s r i f e h o l i t s o e t t r l u l i r r d t w e o o e r g u t p a i h e s o t C e n o F M p ( i a b s r c t FLOW CYTOMETRY IN BIOCHEMICAL ANALYSIS 233

Table 1 SpeciŽc Features, Strategies and Limitations Biochemical assays by  ow cytometry: samples of Functional FCM and probes Because of the unique feature of FCM, i.e., the multipara- metric examination (and physical separation) of single cells or Type of biological samples particles at very fast rate, this particular technique of biochem- Pluricellular organisms Isolated nuclei ² ² ical analysis has evident advantages over other conventional Cell spheroids Subcellular elements methodologies. Thus, the large number of cells analyzed and ² Hybridomas ² Chromosomes the instrumental settings of current cytometers provide multi- ² Cell fusions ² Liposomes ple strategies to obtain primary information, and allow a large ² Human cells ² Mallory bodies number of general applications, as Table 3 attempts to cover. ² Animal cells ² Amyloid plaque Ž bers ² ² From a practical point of view, the main assets of FCM can be Plant protoplasts Membrane fractions summarized as follows: ² Prokaryotic cells ² Viral particles Multiparametric Data Acquisition. Most standard biochem- ² Yeasts ² Soluble antigens1 ical procedures determine a single parameter per assay and are ² Microalgae ² DNA sequences1 ² ² not sensitive enough for single-cell analysis. FCM instruments Type of  uorochromes and  uorescent markers allow routinely two morphology-related parameters (forward- Fluorochromes reacting Fluorescent pH indicators and side-light scatter) and 3–5  uorescence signals per single ² ² with speciŽ c chemical Fluorescent ion chelators particle. In this way, in a single-tube assay, one or more param- ² groups Membrane-potential eters may be used to identify and select (“gated analysis”) cell ² Fluorochrome pairs for sensitive distribution subsets in heterogeneous populations (e.g., live, apoptotic, or ² resonance energy transfer  uorescent dyes necrotic cells; cells of different origin or lineage; cells in dif- Fluorescent antibodies Fluorogenic substrates of ferent cell cycle stage and so on), whereas other signals may ² ² Fluorescent lectins intracellular enzymes be assigned to analyze speciŽ c structures or functions in these ² Fluorescent nucleic acid Fluorescent macromolecules selected populations. An example of this concept is illustrated ² ² sequences Fluorescent synthetic in the single-tube assay shown in Fig. 2. The number of avail- ² Fluorescent lipids particles able parameters per single cell increases when multiple-laser cy- ² Endogenous  uorescent tometers are used. Obviously, the analysis of multiple aliquots ² molecules per sample allows to expand indeŽ nitely the number of param- 1Using  uorescent microspheres as capture reagents and  uores- eters by combining separately  uorescent markers of different cent ligands as reporter molecules. biological properties but similar optical properties. An example of this concept is illustrated in the integrated analysis shown in Fig. 3. This type of FCM analysis (panel analysis) is the hallmark of immunohematology, where typically more than 20  uorescent monoclonal antibodies against epitopes in leukocyte ultimately to regulation of gene expression, cell diferentiation, plasma membrane may be used for typing leukemias and lym- and/or proliferation. phomas (4, 24). FCM is applied successfully to study each step of this vast Multivariate Data Analysis. Due to the hardware and soft- complexity of cellular biochemistry. For most applications, cells ware design of modern cytometers, multiparametric acquisition must be stained with  uorescent markers of deŽ ned optical and is interfaced to multivariate data analysis. In this way, a cell biological properties (Table 1), but FCM takes also advantage population is not described by mere enumeration of the individ- of endogenous  uorochromes related to intracellular functions ual properties measured but by their correlation on a single-cell (1). In this way, as summarized in Table 2, the range of param- basis, thus increasing the discriminating power. Moreover, the eters available for the FCM evaluation of cell biochemistry has possibility of storing FCM data as anuncorrelated data matrix for been extended from broad assessment of cell behaviour to quan- each analyzed cell (“list mode Ž les”) allows one to deŽ ne, if nec- tiŽ cation of single molecules undergoing or regulating speciŽ c essary, new parametric correlations and population selection by biochemical reactions. replaying (off-line) those electronic Ž les. This is an invaluable A signiŽ cant part of FCM studies involves analysis of these tool especially when small or infrequent samples are studied. parameters in relation to cell activation (14, 15) and prolifera- Fast Analysis of Large Number of Live Cells. FCM may be tion (16), cell sensitivity (17, 18) or resistance to drug action performed on a large variety of biological material in different (19), and cell death by apoptosis or necrosis in a wide range of conditions of vitality (e.g., intact fresh cells, Ž xed and/or per- experimental settings (20–22). Although most of these studies meabilized cells), as indicated in Table 3. The use of live cells fall within the scope of basic research, the development of sim- allows one to probe multiple biochemical parameters in mini- ple assays for these parameters has allowed their application to mally perturbed intracellular environments, as well as in near- different clinical situations (3, 23). physiological extracellular conditions. 234 O’CONNOR ET AL.

Table 2 Biochemical assays by  ow cytometry: parameters

Cell surface parameters Cytosolic parameters Nuclear parameters Subcellular elements Membrane integrity General protein DNA content Normal mitochondria Membrane potential Mitochondrial activity RNA content Megamitochondria Membrane recycling Mitochondria content Nuclear total proteins Cis-Golgi vesicles Receptor expression Cytosolic pH Nuclear speciŽ c proteins Trans-Golgi vesicles Receptor interactions Lysosomal pH Chromatin conformation Endosomes Receptor modulation Tyrosine phosphorylation Cyclins and CDks Phagosomes 2 Surface glycoconjugates Cytosolic Ca C Proliferation-related antigens Chloroplasts Ligand binding to surface receptors ROS and NOS DNA synthesis Thylakoids Cell-cell adhesion Enzyme activity: DNA strand breaks Extracellular analytes Membrane  uidity Oxidases DNA oxidation Cholesterol content Dehydrogenases DNA repair Loss of lipid assimetry Esterases Nuclear receptors Permeability to  uorescent probes Proteases Gene expression Membrane peroxidation Transferases Gene reporting Membrane shedding Protein modiŽ cation Endocytosis Free soluble thiols Phagocytosis Glutathione Pynocytosis Protein thiols Ef ux pumps Nonpolar lipids Bacterial cell wall Polar lipids Yeast cell wall Cytoskeletal proteins Granule content

The fast rate of data acquisition and the possibility of ex- which mostly depend on the maintenance of adequate viability amining millions of individual particles in a reasonable time or metabolic capacity of cells and subcellular elements as well allows the detection and accurate analysis of infrequent or rare as avoiding the interference of  uorescent probes with cellular cells, down to 1 event per 108 cells (25). Such a possibility is in functions. contrast with bulk standard  uorimetric determinations in which millions of cells (or their extracts) are analyzed at the same time, FCM Approach to Classic Biochemistry: yielding a single mean concentration value. Flow Cytoenzymology Individual Cell Sorting. Some FCM systems are able to Currently, a wide range of  uorescent substrates or their  uo- separate physically individual cell or particles according to their rogenic precursors are available for FCM analysis (1, 2, 30). On cytometric properties. The most advanced cell sorters are based the other hand,  ow cytometers incorporate time as a parameter on electromagnetic de ection of individual droplets generated by high-frequency vibration of the  ow chamber (1). In such to follow the kinetics of  uorescence variations on the speciŽ c ( ) systems, up to four different subpopulations can be sorted simul- modiŽ cation of substrates 14, 31 . For these reasons,  ow cy- toenzymology (30) appears as a promising application of FCM taneously or, on the other hand, one single cell can be deposited for analysis of metabolism. Flow cytoenzymology is applied to in a given position of a microwell array. Cell sorting allows the a growing number of enzymatic activities in multiple biochem- combination of the intrinsic capabilities of FCM results with ical pathways (32–34) and these studies may have a direct clin- information obtained by image (conventional and confocal mi- ( ) croscopy) and molecular (polymerase chain reaction, in situ hy- ical impact 35, 36 . Thus, they are currently applied to assess leukocyte function, to correlate cell metabolism and malignant bridization) techniques, and provides a preparative tool for rapid capacity in fresh tumor cells, and to evaluate drug metabolism isolation of living rare cells of biochemical relevance, such as and therapy monitorization in pharmacological studies (37). stem cells (26), transfectants (27 ), or hybridomas producing a given antibody (1). It is worth mentioning the contribution of  ow sorting of chromosomes to the sequencing of the human Flow Cytometric Analysis of Isolated genome (28, 29). Subcellular Compartments As indicated in Table 4, there are also critical points and difŽ - The use of  ow cytometers toanalyze functional properties of culties when performing adequate functional analysis by FCM, isolated subcellular particles is less frequent than its application FLOW CYTOMETRY IN BIOCHEMICAL ANALYSIS 235

Table 3 Biochemical assays by  ow cytometry: strategies, information, and applications

Assay strategies Primary information Main general applications a) According to the biological Intensity of expression of multiple IdentiŽ cation/characterization of material: ² parameters within homogeneous ² cells based upon multiple Assays using fresh cells cell populations biochemical parameters ² Assays using Ž xed cells Heterogeneity of expression of multiple Diagnostic applications, including ² Assays using subcellular elements ² parameters in cell subpopulations ² detection of rare pathological cells ² Multiplexed assays Correlation between different parameters Analysis of cell activation, ² ² ² b) According to speciŽ c cell selection: in cell populations including receptor biology and Ratio between multiple parameters signal transduction Nongated assays ² ² Gated assays in single cells Analysis of gene expression, ² Evolution of fast and/or transient ² including gene engineering c) According to assay duration: ² dynamic parameters Analysis of cell cycle and Single end-point assays Evolution of slow and/or sustained ² proliferation-related events ² Sequential end-point assays ² dynamic parameters Analysis of differentiation ² Kinetic assays with unperturbed Detection and analysis of rare ² Flow cytoenzymology ² cells ² cells/particles ² Analysis of cell viability Kinetic assays following cell Correlation with parameters analyzed ² and cell death, including ² stimulation with ligands ² with other techniques following apoptosis and necrosis d) According to data analysis: cell sorting Analysis of microbial biochemistry, On-line analysis (real time) ² including sensitivity to drugs ² Off-line (Listmode analysis) ² Control of biotechnological ² processes, including growth conditions and productivity Environmental biochemistry ² in whole cell studies. However, most current instruments are lular functions and structures in experimental models where a adequately sensitive for subcellular analyses, which have always higher degree of metabolic control can be achieved (Table 1 and been a hallmark of biochemistry. Table 2). Some methodological aspects become critical when analyz- Rhodamine 123 and other membrane-potential (MP)-sensitive ing single subcellular particles by FCM because of their small dyes (15, 31, 38 ) have been used for functional analysis of iso- size, the different permeability or uptake rate of dyes by iso- lated mitochondria, whereas other MP-independent mito- lated organelles, and their usually increased lability. However, chondrial dyes can be applied to determine the mitochondrial FCM analysis of isolated organelles provides insight of subcel- content in whole cells (39). Manipulation of membrane potential in isolated mitochondria induced predictable changes in Rh123 Table 4  uorescence and revealed mitochondrial heterogeneity in liver Biochemical assays by  ow cytometry: difŽ culties cells and heterogeneous responses to physiological and nutritional conditions (40, 41). Isolated mitochondria have been Critical points used also for toxicological and pharmacological studies, which Preparation of single-cell suspensions from adherent cell ² yielded data complementary to those obtained using whole cells populations (42). Maintenance of cell viability along the experimental period FCM has been applied also to analyze the binding of  uo- ² Isolation of subcellular elements from cells and tissues ² rescent lectins to isolated chloroplasts (43) and Golgi vesicles Readjusting conditions for subcellular analysis (44) for the study of their oligosaccharide content. The data thus ² IdentiŽ cation of small cells and particles from background ² obtained may be of relevance to the study of the normal and noise altered mechanisms of glycoprotein maturation and sorting. Adequate access of probes to intracellular sites or processes ² Adequate retention of substrates and probes Cell-Free Cytometry: Quantifying Soluble Analytes ² Noninterference of probes with cell functions FCM is not limited to the analysis of biochemical compo- ² Adequate selection of time-windows for kinetic assays nents in suspensions of cellular or subcellular particles. On the ² Assay calibration for data expression in absolute units ² contrary, a recently developed strategy known as multiplexed 236 O’CONNOR ET AL. , , f s a e n 4 d d d i r C o n a , e e n o L m t H ) t H s o a i r n / F i m p a a t a A o T C c u 1 t b i a g P i g t a s l h I d ) L o d - n a u f n i N F E e n C L t p ( a o i r m f s - , i o B F , s s o t ) e e ) p r s a e H i R e C d n l b ( i e p c E w r H n o p u n s i i f n o s p t C n e e l m o ( a r t a c o c a y B e , i y p s s f t Ž L i H f c S - e i f i m c i p r s o ¹ o c a F i L i r d t - o ( r e s l r u 5 h ¹ u a i p r s t a y l q , s s e  n 5 y p p u c t n y f l r t o 2 l a o l e e i e a o w r , a t e h a h t c o p n t a c n s l s l T T m o l a r a o A u i . . s r o e i d i i t D t r . p r F y - ) f m n . c l a o H . i ) e ) u y y C p p l i w f M ( w a h b e r C d o g t s o u o A e y S s e B r n - s n i r . F g a t m ) F o a o : l e W s i M ( l t F I ( t a d C e ( o y r a c ¹ d e s E i c M h r e e o i C m 4 t p o C t P d i m u r t k f y B d e B R y q s o c u o ( l a h c e o e W h d ) c m e a l t 2 i k n i o c l n t p s H i u L a i a n r a a w o e b , F c r e e l r y h - ) i t t s t L s P i n g e p h M b ( r e s e s o y a m i c u m t C n d t L w r i a n u e F 1 r e r o , c o e i g g r ) a e h a c e o n o ) e e t s p s i b h r I c t c c t e i ( u ) e s d g r t n n t i n - e e d E n e o . e e t t d o ( i ) e s n c u i h l u y t J e s a l w p  ( c g S r s i o e o ) o o n r p S s d n l n r l i w l o B n e s p s s e o u ( d o u e o e f v h h n a i n r t s  s t o a M a g i e n o r n d n i i n d h o g i t H n m d t e i i s n d a t o n i d e 0 k r o r o d a s r a r m n 0 l c o n P g y a a 1 l 1 u l a 5 w e d ) h b L p a f 1 r t o M i e l o n o F m e H e r l / l t ( a h a p A w o e t 2 e L s o - b s f a y t h L n e u n ¹ F e m t i 5 a s o F h o w i 0 C y d y t M t t e f c f C 5 E t y a e C A n o ± o o t c c i - y d C P 7 n t a c s - n Ž F i 3 i n o B o i w o r a 5 l ( s u t C d a o , k 4 m r p i y a v E l 1 u d  c i s D e e a e e t n a L C t a l l i s b L n C e a F u B g u u i r y C m t A r y l a n e g n m i r . a d p 5 d h M o e a 1 v d o - w 1 n e i n e L w n b L l o o L b r e i l , , w a c t c F l ) t ) o h , / X t r e f n o e s n y 2 G s , f e t a b o S ( i d s w v i i L r a i e t e C o o e i t t 0 F a i H I l h n H s t a y 1 w i p i P s o u a p c b r i o u r f n t E r o u 5 o A q o a a H e e f c k 4 P r n r c . s v h I u n a a g n i t i D e n o e ( E s u l l a c n h o e r C l - t y t f i l 1 e o l e t a n s r o p n a c L a n a d a t n i t o n F p m f o u c m c / w a e i a r r o g n e f 2 s r s e f c o n t o e l i f a o n i f L r t e e t p e e d o n F e e L r h a i i m t t s y m c n g w o ¹ i a r e a e a e i , r d t t o s r d d S K 5 a t n m y a i e e i i . . c p a l a t v ) h i v b R e t o p s t i s o e s d i , l n r e h h ) i a e m 0 u w l p w p e n d a n 0 a i o n c t m d a d S m 3 k o n o e H l a i e , e . y s e t t n l h r a . t h S t e c a a ) t t o n r s e s t S c b a y r f e e i e ( A e Ž u h s ( r b o o b o i t r w c i e o d d s e e r s d , m n h i t r l u i ) p e t n c e w p  g i e M a t a e D o r m n c r t e m ( d i r A h r s d e e a - e e s Ž l m o e r y x e b F w a a l ) n c p r i d e 5 c E o s u C t i C t d a s y e c ( s d a n o E t p s r t . n s , C a i o y i i C r ) a o 2 l P x c l e e t p C - b B t a o r p z w e t e - l 5 t i H e o s r o a / x u s l 4 o M p c h u n o i d C l 2 e s s D t g l a m h a i h L r n e n C t o n F w 1 N c ( ( t i F g o a FLOW CYTOMETRY IN BIOCHEMICAL ANALYSIS 237 , t t s s e e g g l s 7 r n a h l n n e a e t 8 i i e t , t c 1 s f s c a w a s t l 3 e o o v e e e l i l 2 l r e v s t c l i e i o h s c A t t o t t i u f o a r a e l s f l a  r o d y o f P p o n e p b o ) o h e - e x r t s p n e A c V Ž i r e ( i o o l s g . n s n e y i s t m t d l i o e e x i a a o l e h ) l e t o n e n p n P t l m a a - y n a b u r l n 2 b i a c i b e i p 6 c l t d n l i e d m o e D n a t i e e n h c e i C c c c ( f m w e k n u i t n o d 0 n e e e d y i i n n 4 s c d b i o s o a 1 n l i e e C d l p s t o l r i i e a C e t e o m d a h g r a w u g d p n v C o s a i  l a t t n l c d i h e g c i 1 o e l e c a n f l 4 t e l i m t t l o s e D n e a r a w a l l b o C l p n o i e a p l t t p o l l g e i - a , a o l t o l n s t v f i u C c i p n y t y n n T c f o c V a I a u i l o a r t f n F n i i p g t i y d s - e e e g x i d l n h ® s e n d u t e i o i t t n e s c y R a S n n h l u i b l ) a t : p a s ) s d f t B d r t ( F e n o e n e n l t . o n e l ) e i n e e p i e a . v c t m E t u ) b i m e a c P r b f a a e - a G l r e r o y s ( - 1 l u a p o n r e s 4 t C p x P r a o t e l a T D e p D e a I G s l e h C c y F A e e ( i s p r n t r e i h a h a e a m t t t r I h l n v i i e c t I g p o I a h i w w r - d s t c n i f e e b y a n o r o h I l l o d i t l o I e u n i b n a e p t d n n i c f o i U g g a a i i s o t r v n . a s x n i e t ) a s g t y e o a n e l e h c a E i c r ( c a s d p D k ( p s t e . e d m x a r ) d e i n c l e o a e F c p r o a e i c ( z t t f a e r r y t e D a n 1 v l l R l i e u – o 4 a e e p s ) t n m k B l D e t C o s a o a h r ( s n t C o l c e t a p . T y i e l n b y P c o . g i n u c c e n o a D g D t l w y f a p a l o o l o A c o i r g o e  s n e t c p h r i n - ) t y n n o i a o s i o h e f c d r p e r w p e y e c p b m t l f o s n Ž e u c l o g t d t i i o g y e t u c i e p l e t n d o e e l n e e i l a a s t t p a p d r p v i r a p a s r i t l t s b h l a e y t p g c e p u h n d m n a t s f i R e e o m t o o ( v n b w ) e h i m i e l o t d n p l e D e l d e n l t o ( f t i v a e a a o i t . o a l c f t n r c i n S p u n Ž s g u a t S m e o i e e g d i t t h e l d g n s n s t n i h a i i s n i s n c n a e e w f o w r n o g h c o o i c i a p i l c d s l b l f l x f o l s o t e o e o a o e f l n d e s c e h e i e s b o l c m T d M o p i a e m l m t f l n . e A r m e P e l o e h u a a p h h 3 h c D s t t x - e t o n w e o d A i E y f e - u h b n b o f l i y m l T . t o t d F i i a d . s 3 r e i x r s e n a t e c t l e o e Ž o n l s p n t i i r i e e x t p t e a i n u l u e h n d t e m d o i g q e i h o d g n t v e d F i c c a a a e a s 238 O’CONNOR ET AL. analysis allows simultaneous quantiŽ cation of multiple analytes 10FIED ISP ES), the Generalitat Valenciana (GV-D-VS-22-148 - in solution. 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