the international agency for research on cancer ( iarc ) has upgraded the cancer classification of formaldehyde in 2006 to now being clearly  carcinogenic to humans  ( group 1 ) .
more recently , formaldehyde has been implicated by the iarc as a causative agent of leukemia as well as nasopharyngeal cancer in humans . the us epa provides a reference dose for chronic oral exposure ( rfd ) of 0.2  mg / kg
bodyweight / day . the who ipcs   has established a tolerable concentration ( tc ) of 2.6  mg / l in ingested products based on animal experiments .
systematic data are currently lacking regarding the formaldehyde content of alcoholic beverages or indeed of most food in general .
estimated that the formaldehyde intake by food may range between 1.5 and 14  mg / person / day , which could , therefore , exceed the rfd in a worst case scenario .
as we had previously conducted in - depth research into acetaldehyde content of alcoholic beverages [ 9 , 10 ] , we were recently asked if we had similar data on formaldehyde and if this compound may pose a risk to consumers in addition to the risk of ethanol .
we had no such data , because formaldehyde can not be analyzed along with the other volatiles ( e.g. , acetaldehyde and methanol ) during a typical gas chromatographic reference method .
it quickly became clear that instrumental techniques such as gc [ 1320 ] , hplc [ 2130 ] , or flow - injection fluorimetric analysis [ 3133 ] , which require extensive sample preparation and derivatization steps , in addition to having costly and complicated instrumental requirements , would not be feasible for the economic and time - efficient survey of a large number of samples .
in contrast , the commonly applied aoac reference method 931.08 , based on the chromotropic acid reaction first described by eegriwe , appeared to be applicable ( figure 1 ) . however , it is a comparatively time - consuming method , which meant that the sample throughput would not have been large ( our aim was to analyze at least 100 samples to provide a valid exposure assessment for consumers ) .
therefore , we had the idea to preselect samples for the aoac procedure by first using a rapid colorimetric screening test for formaldehyde .
a number of these tests are available for screening of aldehydes ( e.g. , fehling 's , tollen 's and schiff 's reagents , see summary by brandl ) , but these classical tests suffer from their low specificity . however , as late as 1970 , a new test using purpald reagent ( 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole , cas # 1750 - 12 - 5 ) was developed , which is remarkably sensitive and specific for aldehydes [ 8 , 37 , 38 ] .
the purpald reaction is based on a condensation of formaldehyde with the reagent to form an aminal , which then reacts under aeration ( facilitated by vigorously shaking of the solution ) to form a purple coloured oxidation product ( figure 2 ) .
the reaction is sensitive for aldehydes , as ketones are oxidized to an uncoloured product [ 8 , 37 ] .
conveniently , the reagents needed for the purpald as well as the chromotropic acid determination are available in ready - to - use test kits ( intended for water or disinfectant analysis ) , and these appear to be usable for testing of alcoholic beverages as well . the major aim of this paper is to evaluate this two - step strategy to provide a survey of formaldehyde in alcoholic beverages .
furthermore , we provide some insight into the structure of the formaldehyde - chromotropic acid chromogen and apply multivariate curve deconvolution techniques to improve the spectrophotometric assay .
1.14678 , based on the chromotropic acid reaction ) , as well as the sulphuric acid ( 9597% ) and absolute ethanol , were purchased from merck ( darmstadt , germany ) .
ortho - phosphoric acid ( 85% ) was obtained from sigma - aldrich ( taufkirchen germany ) .
a formaldehyde standard solution ( 200  mg / l in 40% vol ethanol ) was prepared from a 37% formaldehyde stock solution ( merck , darmstadt , germany ) and confirmed by iodometric titration ( as specified in the application note supplied with the spectroquant test kit ) .
sample temperature was controlled in a dc10-w26 heating circulator bath ( haake , karlsruhe , germany ) .
a vapodest 30 automated distillation device ( c. gerhardt , fabrik und lager chemischer apparate , bonn , germany ) was used for distillation , with condenser cooling at 1c provided by a recirculating chiller b-740 ( bchi , flawil , switzerland ) .
spectrophotometric measurements were performed on a perkin elmer lambda 12 dual beam spectrometer equipped with an automatic cell changer .
the spectra were acquired in a range between 350 and 800  nm at a scanning speed of 120  nm / min with a data interval of 1.0  nm . all measurements were made against ethanol ( 40% vol ) as a blank .
a total of 508 samples of alcoholic beverages ( beer , wine , spirits , and unrecorded alcohol ; see [ 39 , 40 ] for details on unrecorded alcohol ) were analyzed using the purpald method according to the specifications of the test kit .
briefly , 5  ml of the sample were pipetted into a test tube , and five drops of the alkalinization reagent were added to raise the ph to a level above 13 . afterwards , the reaction was started by adding a microspoonful of the purpald reagent .
the solution was then carefully mixed on the vortex mixer . after a reaction time of five minutes , a purple colour indicated a positive result .
uncoloured solutions indicated the absence of any aldehyde ( detection limit at 0.1  mg / l according to the manufacturer ) . only samples that showed a positive purpald reaction needed further analysis .
uncoloured samples ( e.g. , vodka , blanco tequila , fruit spirits ) were used without further preparation .
coloured samples ( e.g. , beer , wine , whiskey ) were distilled according to the aoac method .
the distillation was accomplished using an automated steam distillation device ( see [ 4143 ] for further details on steam distillation in the sample preparation of alcoholic beverages ) . to ensure consistent experimental conditions ,
the system was preheated before every startup , running a blank sample at full steam power ( according to the manufacturer 's instructions ) .
next , 100  ml of sample were brought to 20c and pipetted into a 250  ml kjeldatherm digestion tube . to prevent evaporation
while multiple tests were prepared , the tubes were temporarily sealed using laboratory film . to prepare the receiver , a spatula of crushed ice
was placed in a 50  ml graduated flask before adding 3  ml of sulphuric acid ( 25% ) .
the receiver was then placed underneath the outlet tubing of the distillation device in a beaker filled with ice water to assure cooling during the distillation process ( figure 3 ) . just before clamping the digestion tube into the distillation device , 1  ml of phosphoric acid ( 85% ) was added .
after termination , the flask was tightly sealed , the distillate was brought to 20c in a water bath and filled up to the calibration mark with 40% ethanol .
a 5  ml volume of the sulphuric reagent was pipetted into a test tube , and a microspoonful of the chromotropic acid reagent was dissolved by holding the tube in an ultrasonic bath for approximately 120 seconds .
a 3  ml volume of the sample solution or the distillate was then slowly pipetted into the tube . to start the reaction , the hot tube was closed with a screw cap , and the solution was carefully homogenized using a vortex mixer .
after a reaction time of 10 minutes , the contents of the tube were briefly mixed again .
a 2.5  ml volume of the reaction liquid was transferred into a cuvette for measurement of the absorbance at a wavelength of 565  nm as well as to record a full spectrum .
the calibration was conducted using freshly prepared standards ( 0.5 , 1.0 , 2.0 , 4.0 , 8.0 
mg / l in vodka 40% vol ) treated similarly to the samples . in preliminary experiments with pure standards ,
no interference was noted for the chromotropic acid method even if acetaldehyde was present in large excess .
however while analysing real samples , we noted interferences of other compounds that led to yellowing / browning of the solution . for many samples , the signals created by these matrix compounds were sometimes relatively large and more or less overlapped the formaldehyde peak . for this reason , calibration on a single wavelength ( 565  nm ) is not recommended for formaldehyde quantification in alcoholic beverages .
instead , we evaluated different multivariate techniques such as multivariate curve resolution ( mcr ) or independent component analysis ( ica ) .
these methods are able to extract the target spectrum from a complex matrix and therefore could improve the analytical procedure to determine formaldehyde in alcoholic beverages .
much research in recent years has been done to solve the mixture analysis problem and to extract real spectra and concentration profiles from overlapping spectral data without any a priori assumptions about the composition of the system [ 4448 ] .  
nowadays , the mcr - als ( multivariate curve resolution - alternating least squares ) method developed by tauler [ 46 , 49 , 50 ] is the most well - known and frequently used self - modelling algorithm for spectral deconvolution [ 44 , 51 ] .
results of this method can be considerably improved by applying contraints based on the previous knowledge about the system studied ( e.g. , nonnegativity , closure , unimodality ) .
previous examples in food analysis include the determination of vitamins , food colours , alcohols , and dairy products [ 44 , 5254 ] .
another set of approaches for multivariate analysis is known as  independent component analysis  ( ica ) [ 55 , 56 ] . in some interpretations , ica can be considered as an extension of principal component analysis ( pca ) , the basis of many chemometric methods .
the milca algorithm ( mutual information least dependent component analysis ) is employed in the present study .
this algorithm possesses some unique properties that make it advantageous in comparison with other ica techniques .
milca is based on the search for the least dependent ( in contrast to independent ) mixture components gauged by precise numerical estimates of mutual information   as a measure of signal dependence .
it was found that milca outperforms other specialized chemometric algorithms for spectra decomposition problems and can be used for , for example , the analysis of vitamins [ 47 , 52 ] and human brain samples . the third algorithm we apply is simplisma ( simple - to - use - interactive - self - modelling - mixture - analysis ) , which belongs to the pure variable selection methods .
it finds the most representative row or column profiles for the different compounds in the data set .
when the selectivity conditions are favourable , the row or column can be directly associated with the pure concentration or response profile and the successful resolution of the mixture can be achieved . for mcr calculation we used the software unscrambler x version 10.0.1 ( camo software as , oslo , norway ) .
for the ica calculations , we used matlab v. 7.0 ( the math works , natick , mass , usa ) .
the data set for analysis comprised of the 321 alcoholic beverages tested positive in the purpald assay . to assess the similarities between the resolved and the experimental spectra , we used pearson 's correlation coefficient ( r ) .
hyperchem professional ( hypercube , gainesville , fla , usa ) software package ( v.8.0 ) was used for quantum chemical calculations .
the main goal of all quantum - chemical methods is the solving of the schrdinger equation . in this case
based on the hartree - fok - rutan equation by the self - consistent field ( scf ) method , we applied the semiempirical pm3 ( parametrised model 3 ) method for calculation with full geometry optimization . in most cases
the main approaches of the pm3 method include adiabatic , one - electron , mo lcao ( molecular orbital as a linear combination of atomic orbitals ) and indo ( intermediate neglect of differential overlap ) approximations . for uv - vis spectra calculation
, we used 5 occupied and unoccupied orbitals using the configuration interaction ( ci ) method . for details regarding the calculations ,
c nmr spectra calculations were carried out using chembiodraw 12.0 software ( cambridgesoft , cambridge , uk ) .
chemical shifts are estimated for all hydrogens or carbon atoms for which additivity rules are available .
it also implements models for ethylenes ( cis / trans ) and cyclohexanes ( equatorial / axial ) . in case of c nmr
, it estimates over 95% of the shifts with a mean deviation of 0.29  ppm and standard deviation of 2.8  ppm .
in the first stage of the project , we evaluated the purpald test kit for use with alcoholic beverages ( the test kit was originally intended for disinfectant and rinsing solutions ( e.g. , laundries ) and aqueous solutions ) .
we have spiked vodka samples ( 40% vol ) with different concentrations of formaldehyde and acetaldehyde and not only visually examined the colour reaction but also made a spectrophotometric measurement of the full visible light spectrum .
our results show that the purpald assay is usable with alcoholic solutions , and we were able to confirm the manufacturer 's detection limit of 0.1  mg / l . while it was recognized that the absorption maxima of different aldehydes are too close to allow their differentiation , we are the first to quantitatively record differences in sensitivity between formaldehyde and acetaldehyde ( the assay is approximately 20 times more sensitive for formaldehyde than for acetaldehyde ( figure 4(a ) ) ) . as many alcoholic beverages ( i.e. , especially beer , vodka , or rum ) contain less than 20  mg / l of acetaldehyde , the specificity of this assay is suitable to select formaldehyde - positive samples without an unacceptably high number of false - positives .
although we were able to exclude more than 50% of the original samples as being free of formaldehyde or any other aldehyde detectable with purpald ( figure 5 ) , the assay does not allow us to conclude that a product in fact does contain formaldehyde , so that a more specific confirmation is then needed .
nevertheless , we think that the strategy to screen the samples with purpald is worthwhile , as the assay is very quick , and it allowed us to screen over 500 samples in 3 weeks , which is more than what we had originally intended . to confirm the presence of formaldehyde in the samples , we chose the chromotropic acid procedure , which is suggested by aoac for general use in food analysis .
in contrast to the operating procedure of li et al . , we used an automated steam distillation device , as suggested by steiner and lnzlinger   or liu . in deviation from most previous protocols ,
the commercial test kit   does not require heating of the reagent solution in a water bath ( the heat derived from the dilution of sulphuric acid is sufficient to form the violet dye , as also suggested by steiner et al .   for optimization of the assay ) .
our detection limit ( 0.09  mg / l in vodka ) is similar to that reported in the specifications of the test kit   ( 0.10  mg /
l for 10  mm cells ) , and to that reported by donhauser et al .  
( 0.033  mg / l ) and kleinert and srepel   ( 0.02  mg
of course , techniques using chromatographic separation offer limits in the lower g / l range , but we feel that our limit is sufficient because it is over a factor of 20 lower than the who ipcs tolerable concentration of 2.6  mg / l .
it is also lower than the eu limit for total aldehydes in vodka ( 0.5  g / hl of pure alcohol , which is 2  mg / l for a 40% vol spirit ) .
it is rather surprising that the nature of the chromogen of such a widely used analytical procedure such as the reaction of chromotropic acid with formaldehyde has never been unambiguously proven . according to a hypothesis of georghiou and ho
, the actual product would be the monocationic dibenzoxanthylium structure , while the most often quoted structure ( e.g. , [ 35 , 71 ] ) of the chromogen appears to be the very unlikely para , para - quinoidal structure ( figure 1 ) .
however , as their attempt to crystallize the adduct was unsuccessful , the final structure remains unverified .
to decide , which structure is more likely , we did semiempirical quantum chemical calculations of the uv - vis spectra of the two possible adducts of the reaction ( figure 4(b ) ) .
it has been found that the maxima of the spectral bands for the monocationic dibenzoxanthylium structure are 493 and 563  nm , which is very similar to the experimental values ( first peak around 470490 and second peak around 560580 ) ( figure 6 ) .
the correlation coefficient between the calculated spectrum and the experimental spectral data is relatively high ( r = 0.81 ) . on the other hand
, there is no singlet peak in the region of 560580 nm for the para , para - quinoidal structure , and the correlation is considerably lower ( r = 0.44 ) .
additionally , the spatial situation appears to add to the unlikelihood of the formation of the para , para - quinoidal structure .
calculation of c nmr spectra revealed that the monocationic dibenzoxanthylium structure would have two peaks at 119 and 33  ppm that are comparable with experimental data from the literature ( 119 and 27  ppm ) .
the para , para - quinoidal structure would have a peak at 115  ppm but no signal around 30  ppm .
our uv - vis and c nmr calculations provide evidence that it is most likely that the monocationic dibenzoxanthylium is formed .
our data , therefore , confirm the hypothesis of georghiou . regarding the chromotropic acid reaction , the literature is not consistent regarding its specificity towards formaldehyde .
the original investigation of eegriwe   found no reaction with acetaldehyde or several other aldehydes and further substances .
for determination in alcoholic beverages , it is especially advantageous to have a method in which acetaldehyde , methanol , formic acid , acetic acid , and sugars do not interfere , even if present in the proportion of 10  :  1 or more .
the specificity of the chromotropic assay towards formaldehyde has been experimentally confirmed by several authors [ 63 , 69 , 73 , 74 ] . only ahonen et al .  
detected an interference of acetaldehyde , leading to a lower finding of formaldehyde ( 88% at 1  mg / l of formaldehyde if acetaldehyde was contained in the solution in excess ) . in our experiments ,
no interference was noted even if acetaldehyde was present in large excess ( figure 4(b ) ) .
however , we noted interferences of other compounds that led to yellowing of the solution .
the signals created by these compounds were sometimes relatively large and more or less overlapped the formaldehyde peak . for this reason ,
we do not recommend quantification using calibration on a single wavelength ( 565  nm ) , but instead suggest that a multivariate technique has to be used .
the multivariate models that best describe the investigated system consist of three pure compounds for all chemometric methods used . compared to a set of pure compound spectra ( figure 7(a ) ) , the resolved mcr - als spectra are shown in figure 7(b ) .
it can be seen that one spectrum corresponds to the adduct of formaldehyde - chromotropic acid and the other two belong to interferences .
clearly , the interfering substances strongly absorb at 565  nm and prevent to get accurate results in the single wavelength method .
very similar results were obtained with the two ica algorithms ( figures 7(c ) and 7(d ) ) . to compare the performance of the different algorithms , we calculated the correlation coefficients ( r ) between the experimental spectrum of the adduct of formaldehyde - chromotropic acid reaction and the resolved signals .
the simplisma algorithm gives the best estimation of the spectral signal ( r = 0.97 ) .
however , als ( r = 0.90 ) and milca ( r = 0.95 ) are equally suitable for extracting the pure formaldehyde spectrum . for our final evaluation of the samples ( table 1 )
, we decided to use mcr - als as this is implemented in our standard statistical software package .
the coefficient of variation ( cv ) for spiked vodka ( n = 6 ) was 8.2% at 1 
mg / l , 4.2% at 4  mg / l , and 7.9% at 8 
mg / l ; the recovery for spiked vodka was 101% at 1  mg / l , 99% at 4 
we have not conducted further validation studies in other matrices , but would recommend to study the measurement uncertainty in more detail if results should be used in expert opinions ( e.g. , in legal cases against manufacturers if limits are exceeded ) . in total , formaldehyde was confirmed in 132 samples ( table 1 ) .
the highest incidence was found in tequila ( 83% ) , asian spirits ( 59% ) , grape marc ( 54% ) , and brandy ( 50% ) .
interestingly , formaldehyde levels were not correlated with ethanol ( p = .51 ) , acetaldehyde ( p = .24 ) , or methanol ( p = .94 ) levels that had been determined previously using reference procedures .
we think that the chromotropic acid reaction along with multivariate curve deconvolution is applicable for the purpose to provide a fast and cheap analysis , for example , in the context of high - throughput screening for occurrence and exposure assessment .
we think that only in the case of very high contents of formaldehyde , which would exceed international levels ( e.g. , the who ipcs tolerable concentration ( tc ) of 2.6  mg
/ l   in ingested products ) , additional chromatographic confirmatory analysis is needed prior to taking measures against producers .
in this paper we show how it is possible to quickly survey a large number of samples using a two - step procedure : purpald screening followed by quantitative spectrophotometry using chromotropic acid .
we can confirm the suitability of the chromotropic acid reaction for the determination of formaldehyde in alcoholic beverages , giving results similar to those of li et al . .
it is no wonder that the chromotropic acid method is still widely used , as it is simple and inexpensive .
as we have shown , the procedure can be improved by using multivariate curve deconvolution , which expands its use to matrices that would be normally excluded due to spectral interferences . in our case
, we would go so far to say that the determination of formaldehyde in alcoholic beverages using chromotropic acid is not possible without a chemometric method .
the advantage of the overall procedure is that it is simple , reliable and cheaper than chromatographic methods .  
it should be noted that this approach for the determination of a target compound in a complex matrix with interferences is transferable to other similar spectrophotometric problems .
chemometric approaches can be used for any reaction where the signal of the target compound overlaps with interferences .
we expect that , as these methods become more and more integrated into standard statistical software packages , their use will considerably increase in the future .
our survey of about 500 products showed that only 1.8% of the samples had formaldehyde levels above the who ipcs tolerable concentration .
a 60  kg person would need to consume 0.8  l of alcohol at 14.37  mg / l daily to exceed the us epa rfd of 0.2  mg / kg bodyweight / day , which is extremely unlikely even in this worst - case scenario . while a more detailed population based risk assessment is needed , which also should include other foods , we preliminarily conclude that formaldehyde is unlikely to pose an additional risk for the alcohol drinking population .
 a strategy for analyzing formaldehyde in beer , wine , spirits , and unrecorded alcohol was developed , and 508 samples from worldwide origin were analyzed . in the first step , samples are qualitatively screened using a simple colorimetric test with the purpald reagent , which is extremely sensitive for formaldehyde ( detection limit 0.1  mg 
 / l ) . 
 210 samples ( 41% ) gave a positive purpald reaction . 
 in the second step , formaldehyde in positive samples is confirmed by quantitative spectrophotometry of the chromotropic acid - formaldehyde derivative combined with multivariate curve resolution - alternating least squares ( mcr - als ) . 
 calculation of uv - vis and 13c nmr spectra confirmed the monocationic dibenzoxanthylium structure as the product of the reaction and disproved the widely cited para , para - quinoidal structure . 
 method validation for the spectrophotometric procedure showed a detection limit of 0.09 mg / l and a precision of 4.28.2% cv . in total , 132 samples ( 26% ) contained formaldehyde with an average of 0.27  mg / l ( range 014.4  mg 
 / l ) . 
 the highest incidence occurred in tequila ( 83% ) , asian spirits ( 59% ) , grape marc ( 54% ) , and brandy ( 50% ) . 
 our survey showed that only 9 samples ( 1.8% ) had formaldehyde levels above the who ipcs tolerable concentration of 2.6  mg / l . 
1. Introduction
2. Experimental Section
3. Results and Discussion
4. Conclusions