HPLC Method for Determining the Formaldehyde Content of Beer Weisheng Hu and Dongfeng Wang,1 Laboratory of Food Chemistry and Nutrition, College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China; and Hong Li, China National Research Institute of Food and Fermentation Industries, Beijing 100015, China
It is very important to monitor the formaldehyde content in beer to ensure that the beer conforms to the standards. Much research has resulted in the development of methods for quantitative analysis of formaldehyde in air, food, and drinks (1,5,9,11,17,22, 26,29). These methods can be divided into two kinds: colorimetric and chromatographic. The commonly used color reagents include acetyl acetone and 4-amino hydrazine-5-mercapto-1,2,4triazole (AHMT), but some studies found that these two kinds of color reagents can also react with other aldehydes, such as acetaldehyde (3,27,28). Thus, the assay results of colorimetric methods are susceptible to interference from other coexisting aldehydes in the matrices. Chromatography enables formaldehyde to be isolated from other compounds, so it is preferable if the interference is made obvious. This is often accomplished by derivatization of the formaldehyde to an adduct before injection onto a chromatographic column. Formaldehyde contains an aldehyde group, which makes formaldehyde likely to bind with sulfite or nitrogen-containing compounds (4,6,13). As we know, beer often contains about 10 mg of dioxide sulfide, amino acids, peptide, or proteins per liter. Therefore formaldehyde in beer consists of free species and reversibly bound species. It is thus necessary to completely convert the combined formaldehyde into free species before its derivatization with a derivatizing reagent. The bound formaldehyde species easily become free species under proper acidic and heating conditions. The objective of the present study was to develop an HPLC method for measuring formaldehyde in beer based on derivatization of the formaldehyde to pentafluorophenylhydrazone. The goal was to optimize sample pretreatment and conditions of derivatization.
ABSTRACT J. Am. Soc. Brew. Chem. 73(2):124-129, 2015 Controlling the content of formaldehyde in beer is very important because of formaldehyde’s toxicity. Nevertheless, accurate determination of its content in beer is difficult because of its low concentration in beer and reversible combination with sulfite. A selective and sensitive HPLC method for the determination of formaldehyde in beer, based on the derivatization of formaldehyde and 2,4-dinitrophenylhydrazine (DNPH), is described. Sample pretreatment was as follows: extraction of formaldehyde from beer through steam distillation of a beer sample preceded by adding 20 mL of phosphoric acid (concentration, 200 g/L) into 20 mL of beer and collection of 100 mL of distillate; mixing of 0.5 mL of the distillate and 1 mL of DNPH (concentration,1 g/L) in a 5-mL tube; and derivatizing for 20 min at 60°C. After derivatization, separation of the derivate was achieved using a C18 column (4 × 250 mm, 5 μm) and isocratic elution with 45% acetonitrile. This whole method enabled formaldehyde in beer to be determined with recoveries close to 100%, a repeatability error of less than 1%, and a limit of detection (LOD) of less than 3 μg/L. The overall method was successfully applied to the determination of formaldehyde in marketplace beers. The results showed that the concentration of formaldehyde in Chinese beers ranged from 0.062 to 0.453 mg/L, and the average content of formaldehyde in these beers was 0.134 mg/L. Keywords: Beer, 2,4-Dinitrophenylhydrazine (DNPH), Formaldehyde, HPLC
The interaction of protein and polyphenol is one of the most important causes for nonbiological beer haze (12,25). The effective way to alleviate such haze is to minimize the concentration of either one or both of these components. At present, many commercially available additives are capable of removing excess protein and polyphenol, such as silica gel for protein (15,16) and polyvinylpolypyrrolidone for polyphenol (18–20,23–25). In a previous study, Macey et al found that addition of formaldehyde during the brewing process could give rise to a huge removal of beer polyphenol and an obvious increase in beer colloidal stability (14). Because of the evident effect and low cost of using formaldehyde, it was once used as a stabilizer by some brewers. Nevertheless, epidemiological studies have evaluated the cancer risks associated with formaldehyde exposure (2). Evidence has showed that excessive exposure to formaldehyde can lead to leukemia and cancers of the nasal cavity, nasopharynx, and lung (1,2,10,21). Taking the toxicity of formaldehyde into consideration, many countries have repealed formaldehyde as a food additive and set a maximum limit for the content of formaldehyde in beer. In China, formaldehyde has been removed from the legal food additive list (7), and the concentration of formaldehyde in beer must be below 2 mg/L (8). 1
EXPERIMENTAL Chemicals and Reagents Acetonitrile (HPLC grade) was purchased from Fluka (Buchs, Switzerland). The following compounds with corresponding purities were supplied by the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China): 2,4-dinitrophenylhydrazine (DNPH) (≥99.0%), phosphoric acid (AR grade), formaldehyde (36 to ~38%), and acetic acid (≥99%). Ultrapure water with a resistivity of not less than 18.2 MΩ cm was used throughout the study for all experiments. Samples All the beer samples were produced in China and purchased in the supermarket. Reagent Preparation Derivatizing agent (1,000 mg/L). The derivatizing agent was prepared by dissolving 0.1 g of DNP and 0.5 mL of acetic acid in 100 mL of acetonitrile. Phosphoric acid solution (200 g/L). Phosphoric acid solution was prepared by diluting a concentrated acid in water to a concentration of 200 g/L.
Corresponding author. E-mail:
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http://dx.doi.org/10.1094/ASBCJ-2015-0411-01 © 2015 American Society of Brewing Chemists, Inc.
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Determination of Formaldehyde in Beer Formaldehyde stock solution (1,000 mg/L). The solution was prepared by diluting an appropriate amount of the certified formaldehyde solution. If a certified formaldehyde solution is not available, dilute approximately 2.67 mL of commercially available 37% formaldehyde solution to 1 L with water. The solution should be stored at room temperature in the dark and standardized every 6 months. Formaldehyde work solution (1.0 mg/L). Formaldehyde stock solution (0.10 mL) was diluted to 100 mL with water. It was prepared fresh daily. Analysis of Formaldehyde Using the Optimized Sample Pretreatment Carbonated beer samples were degassed in an ultrasonic bath. Twenty milliliters of degassed sample was added to 20 mL of phosphoric acid (200 g/L) and then transferred into an all-glass steam distiller (Fig. 1). Each sample was distilled using direct steam, and 100 mL of distillate was collected in an ice bath within 15 min. A sample of 20 mL of phosphoric acid of 200 g/L was distilled as the system blank. Each sample was carried out in triplicate. Distillate (1 mL) was pipetted into a 5-mL glass bottle with a polytetrafluoroethylene-lined screw-on lid, to which was added 0.5 mL of DNPH of 1 g/L; the distillate and DNPH were mixed. The glass bottle was placed in an oven for 20 min at 60°C for derivatization. After the derivatization, the sample was cooled down to room temperature using tap water and was filtered by a 0.22-μm membrane syringe filter before injection onto the HPLC system for analysis. HPLC Analysis An Agilent HPLC system (Agilent Technologies, Palo Alto, CA, U.S.A.), incorporating a G1315B diode array detector (DAD) and operated by ChemStation, was used in this study. Chromatographic separation was performed using a reversedphase Thermo Lichrospher 100 RP-18 HPLC column (4.6 × 250 mm, 5 μm) at the column temperature of 35°C. The mobile phase was 45% acetonitrile in water, and the flow rate was 1 mL/min. The injection volume was 10 μL. The UV absorbance at 355 nm was monitored with a DAD. Samples were analyzed in triplicate.
Fig. 1. The all-glass distillation apparatus for formaldehyde distillation. 1 = distiller; 2 = steamer, 1,000-mL boiling flask; 3 = condenser, watercooled; 4 = receiver, 100-mL volumetric flask; 5 = inlet for beer; 6 = electric cooker.
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The target compounds were identified by the retention time and UV-visual spectral characteristics and were quantified using the external standard method. Method Validation The developed method was validated for linearity, precision, sensitivity, and accuracy. The linearity was determined using five levels of calibration in triplicate and was evaluated by the squared correlation coefficient (R2). The calibration range was defined depending on the content of formaldehyde expected in the beer samples. The precision of the method was determined from the repeatability. Six individual preparations of one beer sample were injected, and the percent relative standard deviation (%RSD) was calculated for formaldehyde. The accuracy was evaluated at two different concentrations by adding a known amount of the formaldehyde standard to the beer sample and calculating the percent recovery. The sensitivity of the method was determined by establishing the limit of detection (LOD) with signal-to-noise ratio of 3:1 which was estimated by injecting a series of dilute samples with the known concentration. Recovery by the analytical method was studied by spiking a known amount of formaldehyde into a beer and then analyzing both the spiked and unspiked samples in triplicate. RESULTS AND DISCUSSION Interference of Acetaldehyde with Formaldehyde Determined by the Colorimetric Method After formaldehyde solution (0.2 mg/L) was added to acetaldehyde at concentrations of 0, 5, 10, 15, and 20 mg/L, the absorbance of these solutions was determined using AHMT and acetyl acetone as color reagents. AHMT reacts with aldehydes in strong alkaline media to form a colorless intermediate product. This product is oxidized by atmospheric oxygen to give a magentacolored dye, detectable by colorimetry at a wavelength of 550 nm. In the acetyl acetone method, the reaction of formaldehyde and acetyl acetone results in a yellow compound, which has a maximum absorption at 414 nm. The absorbance results are shown in Figure 2. It shows that acetaldehyde also reacted with AHMT and had absorbance at 550 nm. At the same time, it can also be seen that the absorbance of the reaction product of formaldehyde and acetyl acetone remained stable when the dosage of acetaldehyde was not more than 20 mg/L, but the absorbance of formaldehyde
Fig. 2. Effect of acetaldehyde on absorbance of reacted samples using 4-amino hydrazine-5-mercapto-1,2,4-triazole and acetyl acetone as color reagents.
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at 0.2 mg/L was below 0.1, so it can be inferred that the effect of acetaldehyde on the formaldehyde determination using acetyl acetone as color reagent is negligible. Method Development and Optimization We generated the UV absorbance spectrum for the formaldehyde-DNPH adduct using a DAD. The spectrum is seen in Figure 3. The greatest absorption for the formaldehyde-DNPH adduct was at 355 nm. Thus, 355 nm was chosen as the wavelength for quantitative determination of formaldehyde in beer. Figure 4 shows the effect of coexisting SO2 on the determination of formaldehyde by the HPLC method preceded by derivatization of formaldehyde to adduct without steam distillation. From Figure 4, we can see that the peak area of a 0.2 mg/L concentration of formaldehyde was reduced 80% when it and a 30 mg/L concentration of SO2 coexisted. Thus, it can be inferred that the formaldehyde is apt to combine with SO2 into a formaldehyde-sulfite adduct. In order to get free formaldehyde species from a bound formaldehyde species in beer, acidification of beer using a thermostable acid and heating the beer was employed. Phosphoric acid was used as the acidulant because of its nonvolatility and heat stability. Steam distillation is a mild form of heating that can reduce excessive artificial formaldehyde, so steam distillation was used. Through experiments, we found that 20.0 mL of phosphoric acid (200 g/L con-
centration) was suitable for acidifying beer when 20 mL of beer was sampled. The greater the amount of collected distillate, the more complete was the formaldehyde extraction (Fig. 5). Nevertheless, the concentration of formaldehyde in the condensate was lower and lower, and distillation time was longer and longer with the increase in the distillate volume. Figure 5 demonstrates that the recovery of formaldehyde was essentially 100% when the condensate volume reached 100 mL. Taking time-saving and sensitivity into consideration, we decided to collect 100 mL of distillate when 20.0 mL of beer was distilled by steam in this study. Generally speaking, the common chemical reaction matrix is water. As far as the derivatization reaction in this study is concerned, water also can be used as the solvent. However, sedimentation would occur after derivatization due to the poor solubility of the aldehyde-DNPH derivatives in water, and multiple liquidliquid extraction of the derivatives with organic solvents such as chloroform would be indispensable before injection into an HPLC device. Aldehyde-DNPH derivatives have good solubility in the acetonitrile aqueous solution. In order to save extraction operations and simplify the process, we dissolved DNPH in acetonitrile
Fig. 3. Ultraviolet-visible absorbance spectrum of the formaldehyde–2,4dinitrophenylhydrazine adduct. AU = absorbance unit.
Fig. 5. Effect of the increase in distillate volume on the recovery of formaldehyde.
Fig. 4. Effect of coexisting SO2 on the determination of formaldehyde. DNPH = 2,4-dinitrophenylhydrazine.
Fig. 6. Effect of derivatization time on HPLC peak area of the formaldehyde–2,4-dinitrophenylhydrazine (DNPH) adduct.
Determination of Formaldehyde in Beer (containing 0.5% acetic acid), and the reaction system was 0.5 mL of DNPH-acetonitrile solution and 1 mL of distillate. The DNPH reagent is highly reactive toward formaldehyde. The reaction can proceed completely with the reaction time increase. Figure 6 indicates that the derivatization reaction completed after 20 min at 60°C. Therefore the reaction time of 20 min at 60°C was selected. Typical chromatograms showing the elution profile of the DNPH derivatives of formaldehyde in standard solution and beer are shown in Figure 7. Both formaldehyde in standard solution and formaldehyde in beer were well separated. The retention times of residual DNPH, formaldehyde-DNPH, and acetaldehydeDNPH were 4.8, 9.3, and 13.4 min, respectively. The resolution was good, and there was no excessive interference from the other compounds in beer at these retention times.
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sion analysis and calibration range are shown in Table I. The calibration curve was linear over the concentration range studied. The regression equation had an ideal coefficient of determination (R2 = 0.9998), indicating an excellent fit of the formaldehyde content to the linear model within the range studied. TABLE I Calibration Parameters, Repeatability, and Limit of Detection for the Glycerol Analyte
Formaldehyde
Range (mg/L) Linearity (R2) Repeatability (RSDa %) (n = 6) Limit of detection (mg/L) a
Method Validation The calibration curve was established directly using calibration solutions without steam distillation. The range of concentrations of calibration solutions was from 0.01 to 0.5 mg/L, which was equivalent to 0.05 to 2.5 mg/L in beer because the volume of distillate increased by five times after distillation. The range covered the concentrations normally present in beers. The chromatographic peak area showed a significant positive correlation with the concentration of the formaldehyde. The results of the regres-
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0.05 to ~2.5 0.9998 0.91 3.0
Relative standard deviation. TABLE II Recoveries for the Formaldehyde Standard Added to a Beer (n = 3)
Concentration added (mg/L) 0 0.1313 0.2626
Concentration found (mg/L)
Recovery (%)
0.0851 0.2137 0.3474
97.9 99.9
Fig. 7. HPLC chromatograms of 2,4-dinitrophenylhydrazine (DNPH) derivatives of formaldehyde and acetaldehyde in a standard solution (A) and in beer (B). Peak numbers 1, 2, and 3 were identified as DNPH, formaldehyde-DNPH derivative, and acetaldehyde-DNPH derivative, respectively.
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The resulting RSD was below 1.0%. Table I shows that the LOD was 3.0. The LOD value is in a low enough range to determine the formaldehyde in normal beers. The recoveries of the formaldehyde were near 100% (Table II).
of formaldehyde in normal beers measured with the described method was 0.05 to ~0.5 mg/L.
Marketplace Samples Ten commercially available beers were analyzed for formaldehyde by the developed method in this study. Examples of the results are shown in Table III. In all the cases, the range of levels of formaldehyde in Chinese lager beer were 0.062 to 0.453 mg/L, and the average content of formaldehyde in these beers was 0.134 mg/L, far below 0.9 mg/L, which is upper limit for drinking water set by the World Health Organization.
We acknowledge the National Natural Science Foundation of China under No. 31371731, 31302162 for providing financial support.
CONCLUSIONS In this article, we presented a selective and sensitive method for determination of the formaldehyde in beer based on derivatization of formaldehyde with DNPH at 60°C. HPLC with a DAD and isocratic elution were used for the separation of the formaldehyde-DNPH from other aldehydes plus DNPH. The concentration
TABLE III Formaldehyde Levels in Chinese Bottled Lager Beers Beer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Minimum Maximum Mean
Beer type
Original gravity (°P)
Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Nonpasteurized Alcohol-free Light color Light color Light color Nonpasteurized Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Light color Nonpasteurized Light color Light color Nonpasteurized Light color Light color Light color Light color Light color Light color
8 8 9 11 12 9 8 8 9 8 8 11 ≥3.0 11 10.5 10 10 11 10.5 11.5 9 10 10 11 12 12 13 8 9 10 10 10 11 11 11 8 8 9 10 10
Formaldehyde (mg/L) 0.074 0.085 0.123 0.164 0.166 0.301 0.248 0.333 0.248 0.453 0.123 0.085 0.101 0.101 0.105 0.078 0.118 0.130 0.062 0.101 0.116 0.110 0.083 0.090 0.103 0.108 0.154 0.103 0.147 0.119 0.102 0.116 0.074 0.138 0.133 0.066 0.077 0.099 0.121 0.082 0.062 0.453 0.134
ACKNOWLEDGMENTS
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