BACKGROUND
the enzyme phospholipase d  normally catalyses the formation of phosphatidic acid from phosphatidylcholin using water as a substrate.
in the presence of primary alcohols the reaction is diverted to transphosphatidylation and phosphatidyl alcohol is formed instead.
as a consequence, human ethanol consumption leads to formation of phosphatidylethanol  in the tissues.
in  <dig> hansson et al.
published an article on measurements of peth in whole blood extracts from alcoholics  <cit> .
the separation method used was thin layer chromatography  and subsequently peth was quantified by image analyses.
it was demonstrated that peth was measurable in blood from alcoholics several days, up to weeks, after the last alcohol intake.
peth was suggested to be a potential marker of alcohol abuse.
the tlc separation followed by densitometric scanning was time consuming and had a relatively high coefficient of variation.
to overcome these issues, a hplc method optimised for separating peth from other lipids in whole blood extracts was developed.
peth has physiochemical properties that differ considerably from other phospholipids, which make it possible to separate peth as a single peak at safe distances from other peaks.
an evaporative light scattering  detector was used for detecting the lipids  <cit> .
the elsd detector quantifies any solute less volatile than the solvents.
functional groups, fatty acid chain-length or saturation has little or no effect on the detector response.
however, a non-linear detector response following the quantitative principles of elsd complicates the quantification.
the method was optimized for measuring peth in extracts from whole blood but the same performance parameters are also valid for quantification of peth in extracts from organs  <cit> .
in this paper, five methodological improvements of importance for usefulness and practicability are presented.
first, the extraction procedure is simplified, second, it is shown that peth can be analyzed in blood with edta as anticoagulant, third, the limit of quantification  is lowered, fourth, an internal standard  for the method is presented, and fifth, an improved method for calculation of peth over a broad range is introduced.
RESULTS
extraction
blood samples have in previous publications been extracted according to radin  <cit>  with  <dig> volumes of propan-2-ol:hexane, 3: <dig> v/v.
in practice, this means that  <dig> μl blood can be extracted using  <dig> ml tubes.
the use of  <dig> ml tubes is a limiting factor imposed by the standards in routine clinical laboratories.
it was speculated that adding the blood first to propan-2-ol and thereafter adding the hexane, maintaining the same proportion of solvents, would make the extraction procedure more practical without negatively affecting the recovery of peth.
this variation of radin's extraction method  was investigated and compared to the ordinary extraction according to radin .
thus, in type i extractions, blood was added to the premixed solvents, and in type ii extractions, blood was added to propan-2-ol during mixing before hexane was added.
the same proportion  of solvent was used in both extraction methods.
it was found that the stepwise addition of ingredients resulted in 25% higher recovery of peth , four patient samples were extracted in quintuple of each extraction type).
beside of giving a higher recovery of peth, the stepwise addition is easier to perform because less solvent is present in the tube when the blood is added.
the reason for the higher recovery might be that cell membranes are disrupted and the lipid-protein linkages are broken by the propan-2-ol, which makes the phospholipids from the cell membrane more available than when hexane is present from the start.
recovery studies of extraction procedures for peth have not been performed because of problems in incorporating defined amounts of peth in the same way in the membranes as peth occurs in vivo.
internal standard technique
earlier attempts to find an internal standard  for the peth analysis method were not fruitful  <cit> .
internal standard methodology was judged to be an important improvement in quantification methodology because of its potential to correct for experimental variation .
it was speculated that either phospatidylmethanol  or phosphatidylbutanol  might be suitable as internal standard because of their similarity to peth and for not being endogenous lipids.
the test run of pmet and pbut together with peth standard  showed an rf value for pbut at safe distances from both peth and other peaks while pmet eluted at almost the same rf value as carbamazepine, which is a drug that has anticonvulsant properties and is given to many alcoholic patients at hospitalisation.
the dose response of pbut was very similar to that of peth and by inspecting earlier runs of blood extracts from patients it was asserted that no endogenous compounds eluted at the same rf value as pbut.
it was decided to use pbut as internal standard for the peth determinations.
pbut did not interfere with endogenous lipids in blood.
the extraction method was accommodated to internal standard use by adding  <dig>  nmol of pbut to the propan-2-ol in the test-tube for unknown samples before blood and subsequently hexane were added.
because the detector response is non-linear, it is not optimal to use the simple is evaluation technique where the same amount of is is added to the unknown samples and the calibration samples, and where the calibration curve is constructed from the peak area ratios.
a more accurate approach is to evaluate the apparent amount of both is and analyte from separate standard curves and then apply the is correction.
therefore, typical standard curves were prepared by adding both peth and pbut in different amounts  to the propan-2-ol before blood and subsequently hexane were added.
aliquots of blood from abstainer were kept in a -20°c freezer and used for preparing standard curves; one aliquot was thawed for each preparation.
limit of quantification
the original extraction method  <cit>  was developed to obtain good recovery of all lipids in blood.
since peth is the only lipid of interest in the present method, it was investigated if smaller relative volumes for extraction would be adequate, because that would potentially improve the limit of quantification for peth within the constraint of using  <dig> ml tubes.
the extraction efficiency using  <dig>   <dig> and  <dig> μl blood and  <dig> ml extraction solution  was investigated.
whole blood  was extracted by first adding blood to  <dig> ml of propan-2-ol containing is, and then  <dig> ml of hexane, during agitation.
after mixing twice, the samples were centrifuged at  <dig> g for  <dig> min and the supernatants were transferred to new tubes.
the peth amount recovered per volume of blood was found to be independent of the relative volumes of extraction.
the limit of quantification  for the method has previously been determined to  <dig>  nmol of peth  <cit> .
by increasing the amount of blood that can be extracted for a sample by  <dig> times, the loq, expressed in terms of whole blood concentration, was therefore lowered from  <dig>  to  <dig>  μmol/l, without making the extraction procedure more complicated.
the modified extraction procedure was validated by extracting  <dig> patient samples using  <dig>   <dig> and  <dig> μl whole blood and  <dig> ml solvent.
the result of a 2-way anova of log-transformed values of peth blood concentration showed no significant difference  between the three treatment groups.
the peth blood concentration for these patients varied between  <dig>  and  <dig> μmol/l.
comparison of peth values in blood with different anticoagulants added
blood from five alcoholic patients, being treated for alcohol abstinence, was drawn into both heparin and edta tubes.
all patients had reached zero ethanol in expired air.
the peth amount in the samples was analyzed on day one and thereafter the samples were kept refrigerated and analyzed  <dig>   <dig> and  <dig> weeks after sampling.
the peth amount in the samples was not significantly different between heparin and edta blood at any time point .
furthermore, the peth amount in the samples did not change during three weeks of refrigerated storage .
the ability to use edta blood tubes for sampling makes the analyses more user-friendly since whole blood for haematological test is routinely collected in edta tubes that are not centrifuged, while most routine tests run on blood sampled in heparinized tubes use the plasma or serum after centrifugation, which causes a risk for wrong handling of the sample.
in order to study possible peth formation in vitro in refrigerated blood samples containing ethanol, blood from nine ethanol-intoxicated patients was drawn into edta tubes.
samples were also taken for blood ethanol analyses.
the mean blood ethanol concentration was  <dig> mmol/l .
the peth amount in the samples was analyzed on day one and thereafter the samples were kept refrigerated and analyzed  <dig>   <dig> and  <dig> weeks after sampling.
no significant effect of storage time on peth amount was found .
this stability of peth in refrigerated samples with ethanol present has earlier only been investigated for a time period of three days  <cit> .
freezing blood at -20°c with ethanol present has been shown to highly elevate peth levels whereas such samples can be frozen in liquid nitrogen and stored at -80°c without in vitro formation of peth  <cit> .
the latter method is, however, not practical in a routine setting.
it is therefore important that the present investigation has demonstrated that samples can be stored refrigerated for an extended period of time with no in vitro formation of peth, which in a routine laboratory is an advantage.
calibration and sample evaluation
according to the quantitative principles of elsd, the detector response is non-linear.
curve fitting was therefore used to estimate the calibration function for evaluation of unknown samples.
for the present application the detector response is exponentially shaped at lower amounts of peth and gradually becomes linear at higher amounts.
exploiting this observation, a mixed linear-exponential function, y = a + bx + c exp, was tried and found to accurately trace the calibration data over the complete range of detector response.
here, y is the peak area and x the amount of peth.
the function can easily be reduced to a 3-parameter function by forcing it through the origin .
after substitution and rearrangement, the 3-parameter function becomes y = a) + bx.
the reduction to a 3-parameter model that is bound to the origin is appropriate considering that the peak area is zero in the absence of peth.
in the present application further constraints are that the exponential component diminishes with growing x, that y increases monotonically with growing x, and that x and y are not negative.
therefore, the following numerical constraints apply in the present application: a ≤  <dig>  b ≥  <dig>  and d ≤ -b/a.
a standard levenberg-marquardt algorithm  <cit>  was employed for the non-linear fitting procedure, which for numerical stability, was performed on scaled data.
initial estimates of a and b were obtained by ordinary linear least squares regression of y on a + bx, while d was initially set to -b/a .
then, in order to provide further robustness, the initial estimates were perturbed, one at a time, by a factor of two  and the best fit from the seven starting points was used as the final fit.
an error model with weights taken as 1/y was used because variability is roughly proportional to the response.
to estimate the amount of peth in an unknown sample  from the observed peak area , the inverse of the function was calculated numerically by an iterative range-halving method to a suitable precision defined as the absolute difference between observed y and y calculated from the function.
this approach, of course, hinges on the constraint that the function is monotonously increasing with increasing x. typically, the root-mean-square deviation between the fitted calibration function and the calibration data was about 3% over the range  <dig> – <dig> nmol peth .
the range of the curve fitting procedure used in earlier work  <cit>  was limited to one order of magnitude.
evaluation of unknown samples with internal standard
with each batch of samples, a standard curve, evaluated using the mixed linear-exponential calibration function, was run.
for every unknown sample the apparent amount of pbut was read from the standard curve for pbut and the inverse of this value was multiplied with the known amount of pbut added to the sample to give a sample correction factor.
the apparent amount of peth, obtained from the standard curve for peth was then multiplied with this factor to obtain the corrected amount of peth.
the precision of the method was investigated by running duplicate samples at  <dig> different levels of peth  together with a standard curve on  <dig> separate days.
these samples had been frozen in aliquots of  <dig> μl and for each run, one aliquot was thawed and two extractions  were performed as described.
two different persons did the sample preparations, and the sample order was randomised during preparation and in the hplc run.
the within-day and between-day coefficients of variation  were <6% and <12%, respectively.
the precision obtained using the is methodology and the mixed linear-exponential calibration function   compared with linexp without is  and with the previous method  was validated as follows.
samples from  <dig> patients  were extracted in duplicates of each  <dig>   <dig>  and  <dig> μl whole blood and were evaluated according to the three evaluation models .
precision was calculated as the cv% for the  <dig> samples from each patient .
the precision obtained using the is methodology and the linexp  is several-fold better than obtained with linexp without is  or with the previous method .
this clearly demonstrates the improved ruggedness provided by the is methodology due to its potential to correct for experimental variation .
CONCLUSIONS
both heparinized and edta tubes are suitable as blood sampling tubes.
blood samples with and without ethanol present are stable in refrigerator for three weeks.
the extraction studies showed that more blood could be extracted with the same amount of extraction solution without sacrificing recovery, leading to a threefold improvement in the limit of quantification.
the lowered loq makes it possible to detect intake of lower amount of ethanol than previously.
the development of the internal standard methodology and the mixed linear-exponential calibration function considerably improved robustness, precision and measurable concentration range, all of which are important to facilitate routine application.
the analytical method for peth has been made more robust for use in the routine analytical laboratory.

 the enzyme phospholipase d normally catalyses the formation of phosphatidic acid from phosphatidylcholin using water as a substrate. in the presence of primary alcohols the reaction is diverted to transphosphatidylation and phosphatidyl alcohol is formed instead. as a consequence, human ethanol consumption leads to formation of phosphatidylethanol in the tissues. in hansson et al. published an article on measurements of peth in whole blood extracts from alcoholics. the separation method used was thin layer chromatography and subsequently peth was quantified by image analyses. it was demonstrated that peth was measurable in blood from alcoholics several days, up to weeks, after the last alcohol intake. peth was suggested to be a potential marker of alcohol abuse. the tlc separation followed by densitometric scanning was time consuming and had a relatively high coefficient of variation. to overcome these issues, a hplc method optimised for separating peth from other lipids in whole blood extracts was developed. peth has physiochemical properties that differ considerably from other phospholipids, which make it possible to separate peth as a single peak at safe distances from other peaks. an evaporative light scattering detector was used for detecting the lipids. the elsd detector quantifies any solute less volatile than the solvents. functional groups, fatty acid chain-length or saturation has little or no effect on the detector response. however, a non-linear detector response following the quantitative principles of elsd complicates the quantification. the method was optimized for measuring peth in extracts from whole blood but the same performance parameters are also valid for quantification of peth in extracts from organs. in this paper, five methodological improvements of importance for usefulness and practicability are presented. first, the extraction procedure is simplified, second, it is shown that peth can be analyzed in blood with edta as anticoagulant, third, the limit of quantification is lowered, fourth, an internal standard for the method is presented, and fifth, an improved method for calculation of peth over a broad range is introduced.  extraction blood samples have in previous publications been extracted according to radin with volumes of propan-2-ol:hexane, 3: v/v. in practice, this means that μl blood can be extracted using ml tubes. the use of ml tubes is a limiting factor imposed by the standards in routine clinical laboratories. it was speculated that adding the blood first to propan-2-ol and thereafter adding the hexane, maintaining the same proportion of solvents, would make the extraction procedure more practical without negatively affecting the recovery of peth. this variation of radin's extraction method was investigated and compared to the ordinary extraction according to radin. thus, in type i extractions, blood was added to the premixed solvents, and in type ii extractions, blood was added to propan-2-ol during mixing before hexane was added. the same proportion of solvent was used in both extraction methods. it was found that the stepwise addition of ingredients resulted in 25% higher recovery of peth, four patient samples were extracted in quintuple of each extraction type). beside of giving a higher recovery of peth, the stepwise addition is easier to perform because less solvent is present in the tube when the blood is added. the reason for the higher recovery might be that cell membranes are disrupted and the lipid-protein linkages are broken by the propan-2-ol, which makes the phospholipids from the cell membrane more available than when hexane is present from the start. recovery studies of extraction procedures for peth have not been performed because of problems in incorporating defined amounts of peth in the same way in the membranes as peth occurs in vivo. internal standard technique earlier attempts to find an internal standard for the peth analysis method were not fruitful. internal standard methodology was judged to be an important improvement in quantification methodology because of its potential to correct for experimental variation. it was speculated that either phospatidylmethanol or phosphatidylbutanol might be suitable as internal standard because of their similarity to peth and for not being endogenous lipids. the test run of pmet and pbut together with peth standard showed an rf value for pbut at safe distances from both peth and other peaks while pmet eluted at almost the same rf value as carbamazepine, which is a drug that has anticonvulsant properties and is given to many alcoholic patients at hospitalisation. the dose response of pbut was very similar to that of peth and by inspecting earlier runs of blood extracts from patients it was asserted that no endogenous compounds eluted at the same rf value as pbut. it was decided to use pbut as internal standard for the peth determinations. pbut did not interfere with endogenous lipids in blood. the extraction method was accommodated to internal standard use by adding nmol of pbut to the propan-2-ol in the test-tube for unknown samples before blood and subsequently hexane were added. because the detector response is non-linear, it is not optimal to use the simple is evaluation technique where the same amount of is is added to the unknown samples and the calibration samples, and where the calibration curve is constructed from the peak area ratios. a more accurate approach is to evaluate the apparent amount of both is and analyte from separate standard curves and then apply the is correction. therefore, typical standard curves were prepared by adding both peth and pbut in different amounts to the propan-2-ol before blood and subsequently hexane were added. aliquots of blood from abstainer were kept in a -20°c freezer and used for preparing standard curves; one aliquot was thawed for each preparation. limit of quantification the original extraction method was developed to obtain good recovery of all lipids in blood. since peth is the only lipid of interest in the present method, it was investigated if smaller relative volumes for extraction would be adequate, because that would potentially improve the limit of quantification for peth within the constraint of using ml tubes. the extraction efficiency using and μl blood and ml extraction solution was investigated. whole blood was extracted by first adding blood to ml of propan-2-ol containing is, and then ml of hexane, during agitation. after mixing twice, the samples were centrifuged at g for min and the supernatants were transferred to new tubes. the peth amount recovered per volume of blood was found to be independent of the relative volumes of extraction. the limit of quantification for the method has previously been determined to nmol of peth. by increasing the amount of blood that can be extracted for a sample by times, the loq, expressed in terms of whole blood concentration, was therefore lowered from to μmol/l, without making the extraction procedure more complicated. the modified extraction procedure was validated by extracting patient samples using and μl whole blood and ml solvent. the result of a 2-way anova of log-transformed values of peth blood concentration showed no significant difference between the three treatment groups. the peth blood concentration for these patients varied between and μmol/l. comparison of peth values in blood with different anticoagulants added blood from five alcoholic patients, being treated for alcohol abstinence, was drawn into both heparin and edta tubes. all patients had reached zero ethanol in expired air. the peth amount in the samples was analyzed on day one and thereafter the samples were kept refrigerated and analyzed and weeks after sampling. the peth amount in the samples was not significantly different between heparin and edta blood at any time point. furthermore, the peth amount in the samples did not change during three weeks of refrigerated storage. the ability to use edta blood tubes for sampling makes the analyses more user-friendly since whole blood for haematological test is routinely collected in edta tubes that are not centrifuged, while most routine tests run on blood sampled in heparinized tubes use the plasma or serum after centrifugation, which causes a risk for wrong handling of the sample. in order to study possible peth formation in vitro in refrigerated blood samples containing ethanol, blood from nine ethanol-intoxicated patients was drawn into edta tubes. samples were also taken for blood ethanol analyses. the mean blood ethanol concentration was mmol/l. the peth amount in the samples was analyzed on day one and thereafter the samples were kept refrigerated and analyzed and weeks after sampling. no significant effect of storage time on peth amount was found. this stability of peth in refrigerated samples with ethanol present has earlier only been investigated for a time period of three days. freezing blood at -20°c with ethanol present has been shown to highly elevate peth levels whereas such samples can be frozen in liquid nitrogen and stored at -80°c without in vitro formation of peth. the latter method is, however, not practical in a routine setting. it is therefore important that the present investigation has demonstrated that samples can be stored refrigerated for an extended period of time with no in vitro formation of peth, which in a routine laboratory is an advantage. calibration and sample evaluation according to the quantitative principles of elsd, the detector response is non-linear. curve fitting was therefore used to estimate the calibration function for evaluation of unknown samples. for the present application the detector response is exponentially shaped at lower amounts of peth and gradually becomes linear at higher amounts. exploiting this observation, a mixed linear-exponential function, y = a + bx + c exp, was tried and found to accurately trace the calibration data over the complete range of detector response. here, y is the peak area and x the amount of peth. the function can easily be reduced to a 3-parameter function by forcing it through the origin. after substitution and rearrangement, the 3-parameter function becomes y = a) + bx. the reduction to a 3-parameter model that is bound to the origin is appropriate considering that the peak area is zero in the absence of peth. in the present application further constraints are that the exponential component diminishes with growing x, that y increases monotonically with growing x, and that x and y are not negative. therefore, the following numerical constraints apply in the present application: a ≤ b ≥ and d ≤ -b/a. a standard levenberg-marquardt algorithm was employed for the non-linear fitting procedure, which for numerical stability, was performed on scaled data. initial estimates of a and b were obtained by ordinary linear least squares regression of y on a + bx, while d was initially set to -b/a. then, in order to provide further robustness, the initial estimates were perturbed, one at a time, by a factor of two and the best fit from the seven starting points was used as the final fit. an error model with weights taken as 1/y was used because variability is roughly proportional to the response. to estimate the amount of peth in an unknown sample from the observed peak area, the inverse of the function was calculated numerically by an iterative range-halving method to a suitable precision defined as the absolute difference between observed y and y calculated from the function. this approach, of course, hinges on the constraint that the function is monotonously increasing with increasing x. typically, the root-mean-square deviation between the fitted calibration function and the calibration data was about 3% over the range – nmol peth. the range of the curve fitting procedure used in earlier work was limited to one order of magnitude. evaluation of unknown samples with internal standard with each batch of samples, a standard curve, evaluated using the mixed linear-exponential calibration function, was run. for every unknown sample the apparent amount of pbut was read from the standard curve for pbut and the inverse of this value was multiplied with the known amount of pbut added to the sample to give a sample correction factor. the apparent amount of peth, obtained from the standard curve for peth was then multiplied with this factor to obtain the corrected amount of peth. the precision of the method was investigated by running duplicate samples at different levels of peth together with a standard curve on separate days. these samples had been frozen in aliquots of μl and for each run, one aliquot was thawed and two extractions were performed as described. two different persons did the sample preparations, and the sample order was randomised during preparation and in the hplc run. the within-day and between-day coefficients of variation were <6% and <12%, respectively. the precision obtained using the is methodology and the mixed linear-exponential calibration function compared with linexp without is and with the previous method was validated as follows. samples from patients were extracted in duplicates of each and μl whole blood and were evaluated according to the three evaluation models. precision was calculated as the cv% for the samples from each patient. the precision obtained using the is methodology and the linexp is several-fold better than obtained with linexp without is or with the previous method. this clearly demonstrates the improved ruggedness provided by the is methodology due to its potential to correct for experimental variation.  both heparinized and edta tubes are suitable as blood sampling tubes. blood samples with and without ethanol present are stable in refrigerator for three weeks. the extraction studies showed that more blood could be extracted with the same amount of extraction solution without sacrificing recovery, leading to a threefold improvement in the limit of quantification. the lowered loq makes it possible to detect intake of lower amount of ethanol than previously. the development of the internal standard methodology and the mixed linear-exponential calibration function considerably improved robustness, precision and measurable concentration range, all of which are important to facilitate routine application. the analytical method for peth has been made more robust for use in the routine analytical laboratory. 
the method for peth analysis includes lipid extraction of whole blood, a one-hour hplc separation of lipids and elsd detection of peth. the peth content in blood samples is stable for three weeks in the refrigerator. it is further demonstrated that equal test results are obtained with blood collected in standard test tubes with edta as with the previously used heparinized test tubes. methodological improvements are presented which comprise a simpler extraction procedure, the use of phosphatidylbutanol as internal standard and a new algorithm for evaluation of unknown samples. phosphatidylethanol is an abnormal phospholipid formed slowly in cell membranes by a transphosphatidylation reaction from phosphatidylcholine in the presence of ethanol and catalyzed by the enzyme phospholipase d. peth in blood is a promising new marker of ethanol abuse depending on the high specificity and sensitivity of this marker. none of the biological markers used in clinical routine at the present time are sensitive and specific enough for the diagnosis of alcohol abuse.
 phosphatidylethanol is an abnormal phospholipid formed slowly in cell membranes by a transphosphatidylation reaction from phosphatidylcholine in the presence of ethanol and catalyzed by the enzyme phospholipase d. peth in blood is a promising new marker of ethanol abuse depending on the high specificity and sensitivity of this marker. none of the biological markers used in clinical routine at the present time are sensitive and specific enough for the diagnosis of alcohol abuse. the method for peth analysis includes lipid extraction of whole blood, a one-hour hplc separation of lipids and elsd detection of peth.  methodological improvements are presented which comprise a simpler extraction procedure, the use of phosphatidylbutanol as internal standard and a new algorithm for evaluation of unknown samples. it is further demonstrated that equal test results are obtained with blood collected in standard test tubes with edta as with the previously used heparinized test tubes. the peth content in blood samples is stable for three weeks in the refrigerator.  methodological changes make the method more suitable for routine laboratory use, lower the limit of quantification and improve precision. 