Screening check test to confirm the relative reactivity and applicability of 2,4‐dinitrophenylhydrazine impregnated‐filters for formaldehyde on other compounds

Abstract Objectives A simple check test method was designed to confirm whether a 2,4‐dinitrophenylhydrazine (DNPH) filter for formaldehyde can be used to measure other compounds. Methods Sample mixtures containing the same concentrations of formaldehyde, acetaldehyde, and acetone were spiked to the DNPH‐filter, extracted, and then measured using high performance liquid chromatography with photodiode array detector (HPLC‐PDA). The amounts of DNPH‐derivatives versus the amounts of spiked samples were then plotted. Results When the amount of DNPH << the total amount of spiked samples, the amount of DNPH‐derivatives was formaldehyde > acetaldehyde >> acetone. This order corresponded to the relative rate constants for the reaction. Therefore, this study confirmed that acetone was not collected at the formaldehyde sampling rate. Conclusions This check test easily measured the reaction rate order and can be used as a simple test to determine whether other samples can be measured by the analytical methods used for the specified sample.


| INTRODUCTION
Formaldehyde is carcinogenic to humans; therefore, its presence in workplace air needs to be monitored to determine if exposure controls are adequate. 1 To sample air within the workplace, 2,4-dinitrophenylhydrazine (DNPH)-impregnated silica gel (or filters) are generally employed as a collection and derivatization agent, for both the passive and active sampling of formaldehyde. [2][3][4] 2,4-dinitrophenylhydrazine has been also used for the sample analysis of other aldehyde and ketone because DNPH reacts not only with formaldehyde but also other aldehyde and ketones to produce hydrazone. 5 The DNPH on the filter reacts very quickly with compounds in the air.
Formaldehyde is not the only hazardous compound that needs to be measured in workplaces. Generally, it is preferable if many hazardous target compounds can be measured by fewer analytical methods than the number of target compounds. That is, in the case of hazardous compounds in workplaces where formaldehyde needs to be monitored, it would be best if many compounds could be measured by the DNPH method. However, the reaction rates of hazardous compounds with DNPH are different, depending on the compounds. 6 Furthermore, slight differences in the derivatization conditions can affect the reactivity and yield. 7 Therefore, it would be desirable if the DNPH method for formaldehyde could also be used for the other compounds. It is therefore necessary to confirm whether these other compounds can be measured by the DNPH analytical method.
The reaction rate and relative reaction rate have both been used to evaluate the reactions of various compounds. [8][9][10][11][12][13][14] These methods for reaction rates could be used to confirm whether other compounds are able to be measured by the analytical methods. We reported the relative rate constants for the DNPH derivatization reactions of formaldehyde, acetone, and acetaldehyde, by measuring the DNPH filter spiked with the sample. 15 Using this previously reported reaction rate method 15 is easier than preparing and measuring a sample of accurate concentration into air, but it requires the measurement of many samples to confirm the accuracy of the measurement. A simple method that can quickly determine the possibility of measuring other compounds using the existing method (for example, DNPH-formaldehyde method) is desired; however, no such simple method has yet been proposed or developed.
In this study, a simple test to check the order of reaction rates with DNPH was studied. Furthermore, the feasibility of this method was tested by spiking the mixture sample solution to the DNPH filter.
All the water used in the experiment was purified with Direct-Q UV3 (Merck Millipore and Sigma-Aldrich, Japan Headquarters, Japan).

| Preparation of the extraction sample of the DNPH-impregnated filer spiked with samples
Formaldehyde, acetaldehyde, and acetone were mixed with a molar concentration proportion of 1:1:1. Each compound concentration of the mixed solution was 0.03-3 mol/20 µl (Total amount: 0.09-9 mol/20 µl). And 0.03-3 mol/20 µl of formaldehyde, acetaldehyde, and acetone solution were prepared. With the light off, the sample solutions and the DNPH-impregnated filters (quarter DNPH filter) were heated for 30 min at 25°C, using a heat block in test tubes that were sealed with polytetrafluoroethylene (PTFE)-lined screw caps. With the lights remaining off, these sample solutions (5 μl) were spiked with capillary micro pipets into the DNPH filters in the test tubes, and 0.75 ml of acetonitrile per quarter DNPH filter was added to each test tube (Reaction time (derivatization time): 0 min, which means immediately the sample on the filter was extracted.). These test tubes were then sealed with PTFE-lined screw caps, vortexed, and then ultrasonicated for 10 min. These solutions were used as HPLC samples following filtrations using membrane filters. The amount of DNPH was measured by the amount of DNPH-formaldehyde, which was spiked as formaldehyde (0-135 μmol) onto the DNPH filter to be derivatized. The amount of DNPH-formaldehyde detected was 4-5 μmol/filter (DNPH < total samples, DNPH = total DNPH-samples). Figure 1(A) shows the difference in the amount of each DNPH derivatization when the mixture solutions were spiked to the DNPH filter. When the total amounts of samples (formaldehyde + acetaldehyde + acetone) in the mixture solution was <3 μmol << DNPH (when DNPH < total samples, DNPH = total DNPH-samples), i.e., each sample amount in the mixture solution was <1 μmol (Figure 1(A)), there was little difference in the derivatization ratios among the compounds. However, when the amount of each sample in the mixture solution was 3 μmol, and the total amounts of samples (formaldehyde + acetaldehyde + acetone) >> DNPH, the derivatization ratios among the compounds were quite different. The order of the amounts of the DNPH-derivatives was formaldehyde > acetaldehyde >> acetone. However, the experimental conditions were such that the spiked molarity of formaldehyde, acetaldehyde, and acetone to the DNPH filter were approximately the same, and DNPH generally reacted with the aldehydes and the ketones. The yields of derivatization reactions were different, despite these conditions. This was due to the differences in the reaction rates. Acetone has been reported to react with DNPH slower than formaldehyde. 6 In a previous study, we estimated that the order of the relative reaction rate constants of formaldehyde, acetaldehyde, and acetone on the filter was formaldehyde > acetaldehyde >> acetone. 15 This order was the same as the order of the amounts of the DNPH-derivatized samples when 3 μmol of each sample was spiked onto the filter. Furthermore, regarding the amounts of DNPH-derivatives when the amount of DNPH-formaldehyde was more than the amount of DNPH-sample, the DNPH reaction rate with formaldehyde > the DNPH reaction rate with the other sample. This shows that when the amount of DNPHderivatives equaled the amount of spiked samples, all the spiked sample was derivatized with DNPH. That is, when the derivatization with DNPH was a competitive reaction, and DNPH < the total spiked samples that can react with DNPH, the order of the derivatization amounts was the same as the order of the reaction rates. Furthermore, the difference in derivatization rates at DNPH < total spiked samples was clearer than at DNPH > total spiked samples. This was because all samples spiked with less than the DNPH amounts could be derivatized, whereas a portion of the samples spiked with more than the DNPH amounts could not be derivatized. Figure 1(B) shows the amount of DNPH-formaldehyde and DNPH-acetaldehyde when formaldehyde solution and acetaldehyde solution were spiked to each DNPH filter. All acetaldehyde and formaldehyde were derivatized with DNPH, therefore no difference was found in the reaction rate of acetaldehyde and formaldehyde.

| RESULT AND DISCUSSION
From these results, graphs were obtained by plotting the peak area, or the amount of substances of the DNPHderivatives that were extracted from the filter, on the Yaxis; the total amounts of samples spiked to the filter was plotted as the X-axis. These graphs can be used as a simple check test of the reaction rate comparison. They can also be used to check whether the DNPH filter can be used for other samples. The check test method is as follows: in the first the sample mixture is prepared; it contains the same concentrations of each sample. The second step is obtaining the data, such as that displayed in Figure 1(A). The third step is checking the order of the amounts of the DNPH-derivatives for DNPH < the total amount of spiked sample. If the amount of the DNPH-derivative is equal to the DNPH-other derivatives, a higher concentration sample mixture is spiked, the order of the DNPH-derivatives is checked (Figure 2). Through this method, the reaction rates of the samples were determined to be in the following order: formaldehyde > acetaldehyde >> acetone, and acetone did not react at the sampling rate of formaldehyde. In addition, this screening method can also visually clarify the order of reaction rates for samples with little difference in reaction rates, such as acetaldehyde and formaldehyde ( Figure 1). If there is no difference in the amount of derivatization when the highest concentration sample were added, it will be necessary to be developed a reaction rate measuring device that can control the conditions with high accuracy.

| CONCLUSIONS
To develop a check test, sample mixtures containing the same concentrations of formaldehyde, acetaldehyde, and acetone were spiked to a DNPH-filter. They were then extracted, measured by HPLC-PDA, and the amounts of DNPH-derivatives versus the total amounts of the spiked samples were plotted.
This revealed that the amounts of DNPH-acetaldehyde and DNPH-acetone were not the same as that of DNPHformaldehyde, and the order of the reaction rates was formaldehyde > acetaldehyde >> acetone.
These results showed that this check test can be confirmed that acetone is not collected at the sampling rate of formaldehyde. This check test (using the graph (Figure 1(A)) enables the easy measurement of the orders of reaction rates and the reactivity of other samples with DNPH (sampler). Furthermore, this check test enables the easy measurement of the slightly difference reaction rate order between samples. The test can also confirm, such as acetone measurement with DNPH sampler for formaldehyde, whether other samples can be measured by the analytical methods for the target sample.