# Sampling and Analytical Method for Alpha-Dicarbonyl Flavoring Compounds via Derivatization witho-Phenylenediamine and Analysis Using GC-NPD

Authors: Stephanie M. Pendergrass; Jeffrey A. Cooper
Journal: Scientifica
 (2016)

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## Abstract

A novel methodology is described for the sampling and analysis of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione. These analytes were collected ono-phenylenediamine-treated silica gel tubes and quantitatively recovered as the corresponding quinoxaline derivatives. After derivatization, the sorbent was desorbed in 3 mL of ethanol solvent and analyzed using gas chromatography/nitrogen-phosphorous detection (GC/NPD). The limits of detection (LOD) achieved for each analyte were determined to be in the range of 5–10 nanograms/sample. Evaluation of the on-tube derivatization procedure indicated that it is unaffected by humidities ranging from 20% to 80% and that the derivatization procedure was quantitative for analyte concentrations ranging from 0.1 μg to approximately 500 μg per sample. Storage stability studies indicated that the derivatives were stable for 30 days when stored at both ambient and refrigerated temperatures. Additional studies showed that the quinoxaline derivatives were quantitatively recovered when sampling up to a total volume of 72 L at a sampling rate of 50 cc/min. This method will be important to evaluate and monitor worker exposures in the food and flavoring industry. Samples can be collected over an 8-hour shift with up to 288 L total volume collected regardless of time, sampling rate, and/or the effects of humidity.


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## Body

## 1. Introduction

In August 2000, the National Institute for Occupational Safety and Health (NIOSH) received a request for technical assistance (HETA # 00-0401) in an investigation of severe obstructive lung disease (bronchiolitis obliterans) in former workers of a microwave popcorn plant in Missouri [1]. NIOSH was asked to investigate a cluster of past and present employees experiencing severe respiratory symptoms after working in microwave popcorn processing facilities over a period of 3 months to 3 years [2]. A NIOSH medical and environmental survey at the plant in November 2000 demonstrated a strong exposure-response relationship between quantities of estimated cumulative exposure todiacetyl (a volatile butter flavoring chemical contaminating the air in the plant) and the frequency of airway obstruction on spirometry tests [1].NIOSH method 2557, an air sampling method that uses Anasorb Carbon Molecular Sieve (CMS) sorbent tubes, was developed based on an urgent need for a method to collect and quantitate exposures and evaluate subsequent engineering control effectiveness [3]. This method was used extensively in the field for a number of years. Subsequent field evaluation work suggested a tendency of NIOSH method 2557 to underestimate the true concentration of diacetyl in air [4]. Additional laboratory studies identified that this method had reduced recoveries when samples were collected in moderate-to-high humidity environments. A NIOSH laboratory-based study and a chamber study with generated atmospheres established a correction method for previously collected data with the initial NIOSH method [5]. Concurrently, the Occupational Safety and Health Administration (OSHA) developed method PV2118 that collected diacetyl on a silica gel sorbent. While the method exhibited good storage stability for diacetyl, it had limitations in sampling time/volume because of the collection of water during air sampling [6].In an effort to address the humidity concerns encountered by the NIOSH and OSHA methods, OSHA developed another method for the collection and analysis of diacetyl on specially dried silica gel tubes (2 tubes in series) [7]. By using the dried silica gel tubes in series, this OSHA method addressed migration issues encountered when a single silica gel tube was used. All of these methods utilize gas chromatography equipped with flame ionization detection (GC/FID) for sample analyses.In 2011, NIOSH published a draft criteria document titled “Criteria for a Recommended Standard: Exposure to Diacetyl and 2,3-Pentanedione” that contained a draft NIOSH Recommended Exposure Limit (REL) of 5 pbb, 8 hr-TWA for diacetyl [8]. The criteria document recommended OSHA method 1012 for sampling diacetyl exposures. This method utilizeso-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) to derivatize diacetyl followed by analysis using gas chromatography with electron capture detection (GC-ECD) [9]. OSHA method 1012 has limitations in sampling time and capacity due to the potential collection of water during air sampling, as well as an extended derivatization time up to 36 hours.To address the limitations in diacetyl sampling, a research protocol was designed based upon the derivatization of diacetyl (which was subsequently applied to analogous alpha-dicarbonyl compounds) witho-phenylenediamine (o-PDA). Several research groups have documented the conversion of alpha-dicarbonyl compounds into the corresponding quinoxalines usingo-PDA [10–12].Therefore, the focus of this research project was to develop a method for the collection, derivatization, and stabilization of diacetyl and the other alpha-dicarbonyl compounds (2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione) as quinoxaline derivatives.


## 2. Methods

### 2.1. Apparatus

Gas chromatographic (GC) analyses were conducted using a Hewlett Packard Model 5890 Series II GC with a nitrogen/phosphorus detector (NPD) (Agilent Tech., Avondale, PA) equipped with a 30 m RTX-5 fused silica capillary column (0.25 mm ID, 1μm film) (Restek Corp., Bellefonte, PA).Baseline separation and optimal resolution of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione from the excess derivatizing reagent were achieved using the following parameters. The GC oven temperature program was ramped up from 50°C (held 1 min) to 200°C (10°C/min) and held for 2 min. The injection port temperature was set at 240°C, the detector temperature at 300°C, and the carrier gas (helium) to a flow rate of 1.36 mL/min. The injection solvent was ethanol, which was also used as the method desorption solvent. A splitless GC injection port liner was used and 1μL aliquot was injected.


### 2.2. Reagents

Diacetyl (97%, CAS # 431-03-8),o-PDA (99.5%, CAS # 95-54-5), 2,3-dimethylquinoxaline (97%, CAS # 2379-55-7), 2,3-pentanedione (97%, CAS # 600-14-6), 2,3-hexanedione (≥93%, CAS # 3848-24-6), 2,3-heptanedione (≥97%, CAS # 96-04-8), and ethanol (99.5%, CAS # 64-17-5) were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI).Commercially available silica gel sorbent tubes (SKC # 226-183) and specially preparedo-PDA-treated silica gel sorbent tubes (SKC # CPM021109-001) were obtained from SKC, Inc. (Eighty Four, PA). The commercially available silica gel sorbent tubes contain two sections of silica gel (600 mg front section and 600 mg back section). Theo-PDA- (nominally 0.1% by weight) treated silica gel sorbent tubes contain two sections of treated silica gel (520 mg front section, a PUF separator, and 260 mg back section). Ethanol (99.5%, CAS # 64-17-5) was used as the solvent for all spiking solutions and as the eluting solvent. For all sorbent tubes, the front section (A) and back section (B) were desorbed separately in 3 mL of ethanol in autosampler vials (sealed) and placed on a shaker for 90 minutes to facilitate desorption. Analyte spikes, depending on the study, were placed on the front section of the sorbent tube, or onto the initial glass wool plug, or from generated aerosols. For each concentration level evaluated, six samples (N
=
6) were prepared. A Teflon magnetic stir bar (12.7 mm × 7.9 mm, VWR, Inc.) was placed in each vial. After the desorption period, a portion (1 mL) of each sample was transferred to 2 mL autosampler vials for analysis using GC-NPD (1 μL injection).


### 2.3. Procedures

In order to address the identified limitations of current methods, a number of laboratory evaluations were conducted: (a) determination of LOD and Limit of Quantitation (LOQ), (b) determination of the efficacy of the postsampling derivatization of diacetyl collected on large untreated silica gel tubes, (c) determination of diacetyl, 2,3-pentanedione, and 2,3-hexanedione recovery fromo-PDA-coated silica gel sorbent, (d) determination of the effects of high humidity on the derivatization process, (e) determination of the maximum collection capacity of the coated silica gel sorbent, and (f) determination of analyte storage stability.


#### 2.3.1. LOD/LOQ Determination

Using GC-NPD, eight standards (analyzed in duplicate) were prepared and derivatized on-tube ranging from 2.65 ng/mL to 662.5 ng/mL for diacetyl, from 10 ng/mL to 100.7 ng/mL for 2,3-pentanedione, and from 5 ng/mL to 201.6 ng/mL for 2,3-hexanedione.For LOD and LOQ determination, analytical standards were prepared by serial dilution for diacetyl, 2-pentanedione, and 2,3-hexanedione solutions and 1μL aliquots were spiked directly onto the sorbent tube. After equilibration, the sorbent sections were desorbed in 2 mL of ethanol for 60 minutes [13].


#### 2.3.2. Recovery Study (Untreated Silica Gel Sorbent Tubes)

Initially, untreated silica gel sorbent tubes were prepared for desorption efficiency (DE) studies after the determination of the method LOD/LOQ using GC-NPD. Spikes were prepared at the following levels: 0.0955μg, 9.55 μg, 95.5 μg, 239 μg, and 478 μg.


#### 2.3.3. Recovery Study (o-PDA-Treated Silica Gel Sorbent Tubes)

For the initial desorption efficiency study, where the custom-made ando-PDA-treated silica gel tubes (SKC # CPM021109-001) containing 0.1% o-PDA by weight were used, the desorption efficiencies for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated. Spikes were prepared ranging from approximately 0.1 μg to 500 μg (10 to 100 μg for 2,3-heptanedione) and are listed in Table 1. The ensuing sample preparation and analyses were the same as described in the previous section.Table 1
Preparation of analyte stock solutions and spiking volumes for recovery studies.
Analyte
Spiking level (μg)
Sample volume spiked (μL)
Amount of analyte (μg)
Diacetyl
0.1
0.5
0.0955
10
50
9.55
100
10
95.5
250
25
239
500
50
478
2,3-Pentanedione
0.1
0.5
0.0959
10
50
9.59
100
10
95.9
250
25
240
500
50
480
2,3-Hexanedione
0.1
0.5
0.117
10
50
11.7
100
10
117
250
25
292
500
50
584


#### 2.3.4. Low-Level Recovery Studies

To further define the lower sample recovery limits, a low-level recovery study (0.1 to 1μg) was conducted for each analyte. Using the custom-made, unwashed, and driedo-PDA-treated silica gel tubes (SKC # CPM021109-001), diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated.


#### 2.3.5. Studies of the Effect of Humidity on Recovery

To evaluate the effects of relative humidity on sample collection and recovery, the treated sorbent tubes were placed on an air sampling manifold (Miller-Nelson Flow Temperature Humidity Control System, Model HCS-401) and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was spiked with a solution containing diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at multiple levels ranging from 0.1μg to 500 μg (100 μg for 2,3-heptanedione since it was a minor component in all samples). The tubes were allowed to draw laboratory air for two minutes (50 cc/min) to volatilize the analytes of interest before being connected to a Miller-Nelson atmosphere generator. Humidity-controlled air (20%, 50%, and 80%) was sampled for 240 minutes resulting in a total volume of 12 L. The tubes were then refrigerated overnight. To determine whether breakthrough or migration had occurred during sampling, the sorbent from the individual sections of the tubes was removed and placed into individual 4 mL amber colored desorption vials required to prevent UV degradation of samples.


#### 2.3.6. Capacity Studies

The initial collection capacity study was conducted to evaluate the effects of relative humidity on recovery. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was then spiked with a mixture containing diacetyl, 2,3-pentanedione, and 2,3-hexanedione at concentrations of 1 μg and 500 μg. Spikes were made on the glass wool preceding the sorbent and humid air was pulled through the tubes.The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20% and 80%). Humidity-controlled air was sampled for total volumes ranging from 3 L to 24 L (60 to 480 minutes). Sample preparation and analyses were conducted under the parameters previously described.In an effort to evaluate the effect of an increased sampling rate (200 cc/min) and maximize sampling volumes collected, a more in-depth capacity study was conducted. In this study, 2,3-heptanedione was added as an analyte due to its continued presence as a minor component in alpha-dicarbonyl based flavoring compounds. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 200 cc/min. Each tube was then spiked on glass wool at the front section with a solution of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at 2 levels (N
=
3): 0.5 μg and 100 μg. The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20%, 50%, and 80%).Humidity-controlled air was sampled for total volumes ranging from 96 L to 288 L (480 to 1440 minutes). Tubes were collected from each volume sampled and placed in refrigerated storage overnight. Sample preparation and analyses were conducted under the parameters previously described.


#### 2.3.7. Storage Stability Studies

To evaluate sample stability [13], custom-madeo-PDA-treated silica gel tubes were spiked with 0.6 μg each of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione as shown in Table 2. Six sorbent tubes were analyzed after 1, 7, 14, and 30 days. Separate sets of samples were analyzed after storage under ambient and refrigerated storage conditions. Sample preparation and analyses were conducted under the parameters previously described.Table 2
Standard stock spiking solution preparation and spiking volumes for stability studies.
Analyte
Amount neat analytespike (μL)
Final volume (mL)
Final concentration (μg/mL)
Volume spiked (μL)
Amount spiked (μg)
Diacetyl
1
10
98.5
6
0.591
2,3-Pentanedione
1
10
95.9
6
0.575
2,3-Hexanedione (90%)
1
10
84.0
6
0.504
2,3-Heptanedione
1
10
92.0
6
0.552


## 2.1. Apparatus

Gas chromatographic (GC) analyses were conducted using a Hewlett Packard Model 5890 Series II GC with a nitrogen/phosphorus detector (NPD) (Agilent Tech., Avondale, PA) equipped with a 30 m RTX-5 fused silica capillary column (0.25 mm ID, 1μm film) (Restek Corp., Bellefonte, PA).Baseline separation and optimal resolution of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione from the excess derivatizing reagent were achieved using the following parameters. The GC oven temperature program was ramped up from 50°C (held 1 min) to 200°C (10°C/min) and held for 2 min. The injection port temperature was set at 240°C, the detector temperature at 300°C, and the carrier gas (helium) to a flow rate of 1.36 mL/min. The injection solvent was ethanol, which was also used as the method desorption solvent. A splitless GC injection port liner was used and 1μL aliquot was injected.


## 2.2. Reagents

Diacetyl (97%, CAS # 431-03-8),o-PDA (99.5%, CAS # 95-54-5), 2,3-dimethylquinoxaline (97%, CAS # 2379-55-7), 2,3-pentanedione (97%, CAS # 600-14-6), 2,3-hexanedione (≥93%, CAS # 3848-24-6), 2,3-heptanedione (≥97%, CAS # 96-04-8), and ethanol (99.5%, CAS # 64-17-5) were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI).Commercially available silica gel sorbent tubes (SKC # 226-183) and specially preparedo-PDA-treated silica gel sorbent tubes (SKC # CPM021109-001) were obtained from SKC, Inc. (Eighty Four, PA). The commercially available silica gel sorbent tubes contain two sections of silica gel (600 mg front section and 600 mg back section). Theo-PDA- (nominally 0.1% by weight) treated silica gel sorbent tubes contain two sections of treated silica gel (520 mg front section, a PUF separator, and 260 mg back section). Ethanol (99.5%, CAS # 64-17-5) was used as the solvent for all spiking solutions and as the eluting solvent. For all sorbent tubes, the front section (A) and back section (B) were desorbed separately in 3 mL of ethanol in autosampler vials (sealed) and placed on a shaker for 90 minutes to facilitate desorption. Analyte spikes, depending on the study, were placed on the front section of the sorbent tube, or onto the initial glass wool plug, or from generated aerosols. For each concentration level evaluated, six samples (N
=
6) were prepared. A Teflon magnetic stir bar (12.7 mm × 7.9 mm, VWR, Inc.) was placed in each vial. After the desorption period, a portion (1 mL) of each sample was transferred to 2 mL autosampler vials for analysis using GC-NPD (1 μL injection).


## 2.3. Procedures

In order to address the identified limitations of current methods, a number of laboratory evaluations were conducted: (a) determination of LOD and Limit of Quantitation (LOQ), (b) determination of the efficacy of the postsampling derivatization of diacetyl collected on large untreated silica gel tubes, (c) determination of diacetyl, 2,3-pentanedione, and 2,3-hexanedione recovery fromo-PDA-coated silica gel sorbent, (d) determination of the effects of high humidity on the derivatization process, (e) determination of the maximum collection capacity of the coated silica gel sorbent, and (f) determination of analyte storage stability.


### 2.3.1. LOD/LOQ Determination

Using GC-NPD, eight standards (analyzed in duplicate) were prepared and derivatized on-tube ranging from 2.65 ng/mL to 662.5 ng/mL for diacetyl, from 10 ng/mL to 100.7 ng/mL for 2,3-pentanedione, and from 5 ng/mL to 201.6 ng/mL for 2,3-hexanedione.For LOD and LOQ determination, analytical standards were prepared by serial dilution for diacetyl, 2-pentanedione, and 2,3-hexanedione solutions and 1μL aliquots were spiked directly onto the sorbent tube. After equilibration, the sorbent sections were desorbed in 2 mL of ethanol for 60 minutes [13].


### 2.3.2. Recovery Study (Untreated Silica Gel Sorbent Tubes)

Initially, untreated silica gel sorbent tubes were prepared for desorption efficiency (DE) studies after the determination of the method LOD/LOQ using GC-NPD. Spikes were prepared at the following levels: 0.0955μg, 9.55 μg, 95.5 μg, 239 μg, and 478 μg.


### 2.3.3. Recovery Study (o-PDA-Treated Silica Gel Sorbent Tubes)

For the initial desorption efficiency study, where the custom-made ando-PDA-treated silica gel tubes (SKC # CPM021109-001) containing 0.1% o-PDA by weight were used, the desorption efficiencies for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated. Spikes were prepared ranging from approximately 0.1 μg to 500 μg (10 to 100 μg for 2,3-heptanedione) and are listed in Table 1. The ensuing sample preparation and analyses were the same as described in the previous section.Table 1
Preparation of analyte stock solutions and spiking volumes for recovery studies.
Analyte
Spiking level (μg)
Sample volume spiked (μL)
Amount of analyte (μg)
Diacetyl
0.1
0.5
0.0955
10
50
9.55
100
10
95.5
250
25
239
500
50
478
2,3-Pentanedione
0.1
0.5
0.0959
10
50
9.59
100
10
95.9
250
25
240
500
50
480
2,3-Hexanedione
0.1
0.5
0.117
10
50
11.7
100
10
117
250
25
292
500
50
584


### 2.3.4. Low-Level Recovery Studies

To further define the lower sample recovery limits, a low-level recovery study (0.1 to 1μg) was conducted for each analyte. Using the custom-made, unwashed, and driedo-PDA-treated silica gel tubes (SKC # CPM021109-001), diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated.


### 2.3.5. Studies of the Effect of Humidity on Recovery

To evaluate the effects of relative humidity on sample collection and recovery, the treated sorbent tubes were placed on an air sampling manifold (Miller-Nelson Flow Temperature Humidity Control System, Model HCS-401) and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was spiked with a solution containing diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at multiple levels ranging from 0.1μg to 500 μg (100 μg for 2,3-heptanedione since it was a minor component in all samples). The tubes were allowed to draw laboratory air for two minutes (50 cc/min) to volatilize the analytes of interest before being connected to a Miller-Nelson atmosphere generator. Humidity-controlled air (20%, 50%, and 80%) was sampled for 240 minutes resulting in a total volume of 12 L. The tubes were then refrigerated overnight. To determine whether breakthrough or migration had occurred during sampling, the sorbent from the individual sections of the tubes was removed and placed into individual 4 mL amber colored desorption vials required to prevent UV degradation of samples.


### 2.3.6. Capacity Studies

The initial collection capacity study was conducted to evaluate the effects of relative humidity on recovery. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was then spiked with a mixture containing diacetyl, 2,3-pentanedione, and 2,3-hexanedione at concentrations of 1 μg and 500 μg. Spikes were made on the glass wool preceding the sorbent and humid air was pulled through the tubes.The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20% and 80%). Humidity-controlled air was sampled for total volumes ranging from 3 L to 24 L (60 to 480 minutes). Sample preparation and analyses were conducted under the parameters previously described.In an effort to evaluate the effect of an increased sampling rate (200 cc/min) and maximize sampling volumes collected, a more in-depth capacity study was conducted. In this study, 2,3-heptanedione was added as an analyte due to its continued presence as a minor component in alpha-dicarbonyl based flavoring compounds. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 200 cc/min. Each tube was then spiked on glass wool at the front section with a solution of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at 2 levels (N
=
3): 0.5 μg and 100 μg. The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20%, 50%, and 80%).Humidity-controlled air was sampled for total volumes ranging from 96 L to 288 L (480 to 1440 minutes). Tubes were collected from each volume sampled and placed in refrigerated storage overnight. Sample preparation and analyses were conducted under the parameters previously described.


### 2.3.7. Storage Stability Studies

To evaluate sample stability [13], custom-madeo-PDA-treated silica gel tubes were spiked with 0.6 μg each of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione as shown in Table 2. Six sorbent tubes were analyzed after 1, 7, 14, and 30 days. Separate sets of samples were analyzed after storage under ambient and refrigerated storage conditions. Sample preparation and analyses were conducted under the parameters previously described.Table 2
Standard stock spiking solution preparation and spiking volumes for stability studies.
Analyte
Amount neat analytespike (μL)
Final volume (mL)
Final concentration (μg/mL)
Volume spiked (μL)
Amount spiked (μg)
Diacetyl
1
10
98.5
6
0.591
2,3-Pentanedione
1
10
95.9
6
0.575
2,3-Hexanedione (90%)
1
10
84.0
6
0.504
2,3-Heptanedione
1
10
92.0
6
0.552


## 2.3.1. LOD/LOQ Determination

Using GC-NPD, eight standards (analyzed in duplicate) were prepared and derivatized on-tube ranging from 2.65 ng/mL to 662.5 ng/mL for diacetyl, from 10 ng/mL to 100.7 ng/mL for 2,3-pentanedione, and from 5 ng/mL to 201.6 ng/mL for 2,3-hexanedione.For LOD and LOQ determination, analytical standards were prepared by serial dilution for diacetyl, 2-pentanedione, and 2,3-hexanedione solutions and 1μL aliquots were spiked directly onto the sorbent tube. After equilibration, the sorbent sections were desorbed in 2 mL of ethanol for 60 minutes [13].


## 2.3.2. Recovery Study (Untreated Silica Gel Sorbent Tubes)

Initially, untreated silica gel sorbent tubes were prepared for desorption efficiency (DE) studies after the determination of the method LOD/LOQ using GC-NPD. Spikes were prepared at the following levels: 0.0955μg, 9.55 μg, 95.5 μg, 239 μg, and 478 μg.


## 2.3.3. Recovery Study (o-PDA-Treated Silica Gel Sorbent Tubes)

For the initial desorption efficiency study, where the custom-made ando-PDA-treated silica gel tubes (SKC # CPM021109-001) containing 0.1% o-PDA by weight were used, the desorption efficiencies for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated. Spikes were prepared ranging from approximately 0.1 μg to 500 μg (10 to 100 μg for 2,3-heptanedione) and are listed in Table 1. The ensuing sample preparation and analyses were the same as described in the previous section.Table 1
Preparation of analyte stock solutions and spiking volumes for recovery studies.
Analyte
Spiking level (μg)
Sample volume spiked (μL)
Amount of analyte (μg)
Diacetyl
0.1
0.5
0.0955
10
50
9.55
100
10
95.5
250
25
239
500
50
478
2,3-Pentanedione
0.1
0.5
0.0959
10
50
9.59
100
10
95.9
250
25
240
500
50
480
2,3-Hexanedione
0.1
0.5
0.117
10
50
11.7
100
10
117
250
25
292
500
50
584


## 2.3.4. Low-Level Recovery Studies

To further define the lower sample recovery limits, a low-level recovery study (0.1 to 1μg) was conducted for each analyte. Using the custom-made, unwashed, and driedo-PDA-treated silica gel tubes (SKC # CPM021109-001), diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were evaluated.


## 2.3.5. Studies of the Effect of Humidity on Recovery

To evaluate the effects of relative humidity on sample collection and recovery, the treated sorbent tubes were placed on an air sampling manifold (Miller-Nelson Flow Temperature Humidity Control System, Model HCS-401) and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was spiked with a solution containing diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at multiple levels ranging from 0.1μg to 500 μg (100 μg for 2,3-heptanedione since it was a minor component in all samples). The tubes were allowed to draw laboratory air for two minutes (50 cc/min) to volatilize the analytes of interest before being connected to a Miller-Nelson atmosphere generator. Humidity-controlled air (20%, 50%, and 80%) was sampled for 240 minutes resulting in a total volume of 12 L. The tubes were then refrigerated overnight. To determine whether breakthrough or migration had occurred during sampling, the sorbent from the individual sections of the tubes was removed and placed into individual 4 mL amber colored desorption vials required to prevent UV degradation of samples.


## 2.3.6. Capacity Studies

The initial collection capacity study was conducted to evaluate the effects of relative humidity on recovery. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 50 cc/min. Each tube was then spiked with a mixture containing diacetyl, 2,3-pentanedione, and 2,3-hexanedione at concentrations of 1 μg and 500 μg. Spikes were made on the glass wool preceding the sorbent and humid air was pulled through the tubes.The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20% and 80%). Humidity-controlled air was sampled for total volumes ranging from 3 L to 24 L (60 to 480 minutes). Sample preparation and analyses were conducted under the parameters previously described.In an effort to evaluate the effect of an increased sampling rate (200 cc/min) and maximize sampling volumes collected, a more in-depth capacity study was conducted. In this study, 2,3-heptanedione was added as an analyte due to its continued presence as a minor component in alpha-dicarbonyl based flavoring compounds. The custom-madeo-PDA-treated silica gel tubes were placed on the air sampling manifold and the flow rate of the manifold was adjusted to 200 cc/min. Each tube was then spiked on glass wool at the front section with a solution of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione at 2 levels (N
=
3): 0.5 μg and 100 μg. The tubes were allowed to draw laboratory air for two minutes to volatilize the analytes before being connected to an atmosphere generator to produce the humidity-controlled air (20%, 50%, and 80%).Humidity-controlled air was sampled for total volumes ranging from 96 L to 288 L (480 to 1440 minutes). Tubes were collected from each volume sampled and placed in refrigerated storage overnight. Sample preparation and analyses were conducted under the parameters previously described.


## 2.3.7. Storage Stability Studies

To evaluate sample stability [13], custom-madeo-PDA-treated silica gel tubes were spiked with 0.6 μg each of diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione as shown in Table 2. Six sorbent tubes were analyzed after 1, 7, 14, and 30 days. Separate sets of samples were analyzed after storage under ambient and refrigerated storage conditions. Sample preparation and analyses were conducted under the parameters previously described.Table 2
Standard stock spiking solution preparation and spiking volumes for stability studies.
Analyte
Amount neat analytespike (μL)
Final volume (mL)
Final concentration (μg/mL)
Volume spiked (μL)
Amount spiked (μg)
Diacetyl
1
10
98.5
6
0.591
2,3-Pentanedione
1
10
95.9
6
0.575
2,3-Hexanedione (90%)
1
10
84.0
6
0.504
2,3-Heptanedione
1
10
92.0
6
0.552


## 3. Results

### 3.1. LOD/LOQ Determination

As previously described, eight standards (in duplicate) were analyzed using GC-NPD: diacetyl (2.65 to 662.5 ng/mL), 2,3-pentanedione (10 to 100.7 ng/mL), and 2,3-hexanedione (5 to 201.6 ng/mL). The instrumental LOD and LOQ were determined using calibration curves (diacetyl – slope = 904.66, intercept = 15.74, andR
2
=
0.9216; 2,3-pentanedione – slope = 533.19, intercept = 29.8, and R
2
=
0.7918; 2,3-hexanedione – slope = 426.30, intercept = 2.5, and R
2
=
0.9796; and 2,3-heptanedione – slope = 113.28, intercept = 28.95, and R
2
=
0.9716). Results are listed in Table 3.Table 3
Limit of detection (LOD) and Limit of Quantitation (LOQ) for alpha-dicarbonyl compounds.
Analyte
LOD14
LOQ14
Diacetyl
7 ng/mL
23 ng/mL
2,3-Pentanedione
17 ng/mL
58 ng/mL
2,3-Hexanedione
5 ng/mL
15 ng/mL


### 3.2. Recovery Study (Untreated Silica Gel Sorbent Tubes)

On the basis of the initial recovery results achieved when diacetyl was spiked directly on untreated silica gel tubes and desorbed in a solution of 1 mg/mL ofo-PDA, a full scale recovery study was evaluated. Desorption efficiency recoveries for 2,3-dimethylquinoxaline (diacetyl derivative) ranged from 56.3% (0.0955 μg) to 104.3% (478 μg) with an average Relative Standard Deviation (RSD) of 0.039 are listed in Table 4.Table 4
Diacetyl recovery after extraction of spiked sorbent witho-PDA solution.
Spike level (μg)
Average recovery (%)
RSD
0.096
56.3
0.094
9.55
96.4
0.019
95.5
93.0
0.032
239.0
80.1
0.017
478.0
104.3
0.033


### 3.3. Recovery Study (o-PDA-Treated Silica Gel Sorbent Tubes)

The next phase in the method development process for the derivatization of diacetyl, 2,3-pentanedione, and 2,3-hexanedione was to determine the feasibility of collecting and derivatizing the analytes “on-tube” usingo-PDA-coated silica gel sorbent tubes. The initial glass wool plugs were spiked with diacetyl. Ambient air, generated by a Miller-Nelson atmospheric generator, was drawn through the tubes at 0.05 L/min. Desorption efficiencies for the analytes’ derivatization witho-PDA are depicted in Table 5.Table 5
Recovery results for the on-tube derivatization of diacetyl, 2,3-pentanedione, and 2,3-hexanedione.
Analyte
Spike level (μg)
Average recovery (%)
RSD
Diacetyl
0.096
87.0
0.106
9.55
99.6
0.037
95.5
91.4
0.099
239.0
104.0
0.080
478.0
103.0
0.077
2,3-Pentanedione
0.96∗
—∗
—∗
9.59
106.5
0.045
95.9
96.1
0.043
240.0
92.3
0.064
480.0
87.3
0.041
2,3-Hexanedione
0.117
69.7
0.212
11.7
73.5
0.032
117.0
69.6
0.032
292.0
66.8
0.040
584.0
62.5
0.028
∗Data unavailable due to sample loss during analytical preparation.


### 3.4. Low-Level Recovery Studies

As noted earlier in the Methods, after the successful recovery study at levels above 1μg, a low-level recovery study was initiated. The recoveries for diacetyl ranged from 87.2% to 100.7% with an average RSD of 0.073; for 2,3-pentanedione ranged from 94.8% to 120.1% with an average RSD of 0.068; for 2,3-hexanedione ranged from 105.1% to 117.3% with an average RSD of 0.058; and for 2,3-heptanedione ranged from 83.4% to 90.6% with an average RSD of 0.071. Results are listed in Table 6.Table 6
Low level recovery study for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione.
Analyte
Spike level (μg)
Recovery (%)
RSD
Diacetyl
0.099
87.2
0.139
0.591
96.7
0.046
0.985
100.7
0.036
2,3-Pentanedione
0.096
120.1
0.112
0.575
94.8
0.051
0.959
108.8
0.038
2,3-Hexanedione
0.084
105.1
0.043
0.504
117.3
0.056
0.840
105.1
0.043
2,3-Heptanedione
0.092
83.4
0.072
0.552
90.5
0.071
0.920
90.6
0.065


### 3.5. Studies of the Effect of Humidity on Recovery

Due to the negative effects that humidity has on diacetyl recovery discovered during some of the more recent field sampling surveys conducted at food and flavoring sites using NIOSH method 2557 [5], the next progression in our method development effort was to evaluate the effect on sample collection of various levels of humidity when using theo-PDA-coated silica gel sorbent tubes. Spiking levels ranged from approximately 0.1 μg to 500 μg. After sample collection for a period of 240 minutes and a total volume of 12 L, recoveries were determined for each derivatized analyte collected at relative humidities of 20%, 50%, and 80% (actual measured humidity). The results are listed in Table 7.Table 7
Effects of varying humidity levels on analyte recovery.
Analyte
Level (μg)
Relative humidity (%)
Mean recovery
RSD
Diacetyl
0.118
16.7
86.4
0.130
0.118
58.0
101.0
0.146
0.118
80.1
72.5
0.123
0.640
22.6
97.7
0.040
0.640
58.0
111.0
0.098
0.640
80.4
79.3
0.076
0.938
20.7
92.4
0.032
0.938
58.0
115.0
0.121
0.938
78.5
97.8
0.110
9.85
17.9
107.6
0.035
9.85
52.0
100.1
0.076
9.85
79.6
99.6
0.028
98.5
17.9
111.0
0.019
98.5
52.0
100.1
0.076
98.5
49.6
99.6
0.028
246.25
17.9
105.0
0.074
246.25
51.1
101.2
0.087
246.25
80.6
88.9
0.042
492.5
17.9
102.7
0.049
492.5
50.0
108.9
0.037
492.5
81.0
99.5
0.022
2,3-Pentanedione
0.115
16.7
63.9
0.133
0.115
58.0
98.7
0.112
0.115
80.1
73.9
0.123
0.622
22.6
88.4
0.031
0.622
58.0
85.6
0.080
0.622
80.4
76.7
0.074
0.909
20.7
84.9
0.026
0.909
58.0
102.0
0.142
0.909
78.5
98.8
0.137
9.59
21.1
94.8
0.072
9.59
51.3
85.8
0.071
9.59
80.8
64.1
0.107
95.9
21.1
102.7
0.085
95.9
51.4
100.0
0.102
95.9
79.7
46.2
0.060
239.75
21.1
104.9
0.051
239.75
50.7
99.1
0.119
239.75
80.6
73.9
0.110
479.5
21.1
90.2
0.084
479.5
50.4
87.1
0.125
479.5
79.9
80.3
0.112
2,3-Hexanedione
0.101
16.7
120.0
0.195
0.101
58.0
114.3
0.085
0.101
80.1
49.9
0.062
0.546
22.6
108.0
0.038
0.546
58.0
109.0
0.060
0.546
80.4
82.4
0.064
0.799
20.7
103.8
0.034
0.799
58.0
124.0
0.206
0.799
78.5
90.8
0.107
11.68
21.1
67.8
0.055
11.68
51.3
60.4
0.056
11.68
80.8
45.4
0.078
116.75
21.1
66.4
0.051
116.75
51.4
68.6
0.060
116.75
79.7
31.1
0.039
291.88
21.1
66.8
0.028
291.88
50.7
72.1
0.107
291.88
80.6
40.4
0.080
583.75
21.1
59.6
0.057
583.75
50.4
52.5
0.079
583.75
79.9
53.7
0.088
2,3-Heptanedione
0.110
16.7
90.3
0.180
0.110
58.0
69.8
0.142
0.110
80.1
75.9
0.062
0.598
22.6
81.0
0.034
0.598
58.0
88.7
0.066
0.598
80.4
78.4
0.061
0.874
20.7
75.3
0.067
0.874
58.0
108.0
0.195
0.874
78.5
107.0
0.111
9.20
21.5
84.9
0.099
9.20
47.3
84.5
0.053
9.20
80.3
81.9
0.077
92.0
21.5
103.1
0.044
92.0
52.7
92.3
0.030
92.0
83.8
90.2
0.031


### 3.6. Capacity Studies

In the initial collection capacity study, diacetyl, 2,3-pentanedione, and 2,3-hexanedione were sampled on theo-PDA-coated silica gel tubes at two levels (1 μg and 500 μg) for total air collection capacities ranging from 3 L (60 min) to 24 L (480 min). Sampling was conducted at relative humidities of 20% and 80% and the results are depicted in Tables 8 and 9.Table 8
Determination of air sampling capacity ofo-PDA-treated silica gel tubes at 20% relative humidity at 2 concentration levels.
Analyte
Level (μg)
Sampling volume (L)
Mean recovery (%)
RSD
Diacetyl
0.985
3
85.5
0.016
0.985
6
99.9
0.059
0.985
12
91.1
0.058
0.985
18
84.0
0.020
0.985
24
102.9
0.131
490.0
3
91.1
0.038
490.0
6
104.6
0.052
490.0
12
99.0
0.014
490.0
18
91.8
0.019
490.0
24
100.4
0.058
2,3-Pentanedione
0.959
3
105.2
0.056
0.959
6
103.3
0.085
0.959
12
105.8
0.053
0.959
18
85.8
0.016
0.959
24
106.7
0.098
480.0
3
96.3
0.038
480.0
6
97.0
0.045
480.0
12
110.6
0.028
480.0
18
116.6
0.077
480.0
24
102.3
0.039
2,3-Hexanedione
0.841
3
72.7
0.087
0.841
6
91.8
0.077
0.841
12
110.8
0.109
0.841
18
85.0
0.016
0.841
24
92.6
0.114
420.0
3
89.6
0.036
420.0
6
86.4
0.089
420.0
12
90.8
0.059
420.0
18
110.0
0.032
420.0
24
108.5
0.103Table 9
Determination of air sampling capacity ofo-PDA-treated silica gel tubes at 80% relative humidity at 2 concentration levels.
Analyte
Level (μg)
Sampling volume (L)
Mean recovery (%)
RSD
Diacetyl
0.985
3
78.1
0.016
0.985
6
82.1
0.058
0.985
12
95.2
0.097
0.985
18
82.4
0.034
0.985
24
103.6
0.028
490.0
3
97.4
0.047
490.0
6
88.5
0.074
490.0
12
112.7
0.034
490.0
18
100.6
0.018
490.0
24
93.7
0.065
2,3-Pentanedione
0.959
3
100.1
0.030
0.959
6
76.1
0.051
0.959
12
101.2
0.068
0.959
18
97.8
0.048
0.959
24
81.1
0.139
480.0
3
97.8
0.055
480.0
6
92.3
0.085
480.0
12
99.2
0.058
480.0
18
87.1
0.010
480.0
24
85.2
0.071
2,3-Hexanedione
0.841
3
100.7
0.018
0.841
6
69.5
0.077
0.841
12
98.3
0.056
0.841
18
101.4
0.027
0.841
24
80.4
0.141
420.0
3
95.5
0.049
420.0
6
92.5
0.086
420.0
12
87.7
0.053
420.0
18
94.7
0.048
420.0
24
87.6
0.173In an effort to evaluate the effect of an increased sampling rate (200 cc/min) and maximize sampling volumes collected, a more in-depth capacity study was conducted. In this study, 2,3-heptanedione was added as an analyte due to its continued presence as a minor component (contaminant) in alpha-dicarbonyl based flavoring compounds. A more detailed depiction of the recovery data for each analyte is presented in Table10 (20% RH), Table 11 (50% RH), and Table 12 (80% RH). Mean recovery (%) was calculated based on the average recovery of 3 samples evaluated at each volume sampled.Table 10
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 20% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.493
96
480
99.8
0.035
0.493
144
720
110.0
0.040
0.493
216
960
104.0
0.054
0.493
288
1440
108.0
0.043
98.5
96
480
98.3
0.010
98.5
144
720
104.0
0.023
98.5
216
960
100.0
0.038
98.5
288
1440
95.5
0.073
2,3-Pentanedione
0.479
96
480
92.1
0.015
0.479
144
720
83.0
0.046
0.479
216
960
101.0
0.088
0.479
288
1440
109.0
0.181
95.7
96
480
97.8
0.038
95.7
144
720
99.4
0.021
95.7
216
960
103.0
0.156
95.7
288
1440
95.8
0.046
2,3-Hexanedione
0.420
96
480
97.1
0.021
0.420
144
720
102.0
0.036
0.420
216
960
109.0
0.016
0.420
288
1440
106.0
0.061
84.1
96
480
87.9
0.036
84.1
144
720
101.0
0.021
84.1
216
960
99.4
0.032
84.1
288
1440
84.6
0.047
2,3-Heptanedione
0.460
96
480
104.0
0.027
0.460
144
720
101.0
0.065
0.460
216
960
106.0
0.025
0.460
288
1440
108.0
0.052
92.0
96
480
93.9
0.082
92.0
144
720
98.6
0.029
92.0
216
960
104.0
0.024
92.0
288
1440
93.5
0.052Table 11
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 50% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.493
96
480
102.0
0.015
0.493
144
720
92.6
0.024
0.493
216
960
102.0
0.018
0.493
288
1440
87.0
0.026
98.5
96
480
98.8
0.027
98.5
144
720
106.0
0.016
98.5
216
960
101.8
0.020
98.5
288
1440
101.0
0.028
2,3-Pentanedione
0.479
96
480
92.7
0.010
0.479
144
720
77.8
0.054
0.479
216
960
98.5
0.158
0.479
288
1440
88.5
0.009
95.7
96
480
97.8
0.030
95.7
144
720
100.0
0.019
95.7
216
960
110.0
0.024
95.7
288
1440
97.8
0.026
2,3-Hexanedione
0.420
96
480
93.7
0.024
0.420
144
720
86.2
0.021
0.420
216
960
87.9
0.020
0.420
288
1440
71.4
0.018
84.1
96
480
88.0
0.023
84.1
144
720
102.0
0.023
84.1
216
960
103.0
0.026
84.1
288
1440
89.3
0.018
2,3-Heptanedione
0.460
96
480
103.0
0.019
0.460
144
720
89.3
0.026
0.460
216
960
97.8
0.031
0.460
288
1440
98.5
0.041
92.0
96
480
94.1
0.020
92.0
144
720
99.8
0.017
92.0
216
960
105.0
0.011
92.0
288
1440
98.6
0.019Table 12
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 80% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.985
96
480
99.7
0.017
0.985
144
720
101.0
0.110
0.985
216
960
93.7
0.055
0.985
288
1440
84.1
0.114
98.5
96
480
90.5
0.072
98.5
144
720
98.8
0.036
98.5
216
960
93.5
0.057
98.5
288
1440
89.4
0.037
2,3-Pentanedione
0.957
96
480
72.9
0.081
0.957
144
720
112.0
0.055
0.957
216
960
105.0
0.114
0.957
288
1440
99.5
0.054
95.7
96
480
86.0
0.055
95.7
144
720
93.8
0.038
95.7
216
960
55.2
0.054
95.7
288
1440
48.8
0.035
2,3-Hexanedione
0.841
96
480
119.0
0.138
0.841
144
720
122.0
0.062
0.841
216
960
105.0
0.114
0.841
288
1440
44.8
0.031
84.1
96
480
86.2
0.060
84.1
144
720
93.5
0.039
84.1
216
960
60.4
0.053
84.1
288
1440
54.6
0.015
2,3-Heptanedione
0.920
96
480
52.0
0.021
0.920
144
720
104.0
0.089
0.920
216
960
76.0
0.154
0.920
288
1440
57.2
0.055
92.0
96
480
85.5
0.058
92.0
144
720
93.7
0.036
92.0
216
960
57.8
0.050
92.0
288
1440
53.7
0.018


### 3.7. Storage Stability Recoveries

Evaluation of the ambient and refrigerated storage stability recovery results for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione indicates that the derivatized analytes were stable for up to 30 days at the 0.6μg spiking levels. The average storage stability results evaluated for each analyte at 1, 7, 14, and 30 days are reported in Table 13.Table 13
Storage stability conducted under ambient and refrigerated storage conditions.
Day
Avg. recovery (ambient, %)
RSD
Avg. recovery (refrigerated, %)
RSD
Diacetyl
1
96.1
0.068
96.7
0.046
7
93.4
0.048
89.8
0.039
14
98.6
0.017
106.0
0.065
30
103.0
0.039
98.6
0.047
2,3-Pentanedione
1
98.0
0.049
94.8
0.051
7
102.0
0.045
100.0
0.025
14
105.0
0.091
97.1
0.068
30
108.0
0.065
96.8
0.073
2,3-Hexanedione
1
109.8
0.115
117.0
0.056
7
93.9
0.041
110.0
0.054
14
87.0
0.068
99.0
0.040
30
81.4
0.026
99.6
0.053
2,3-Heptanedione
1
89.3
0.109
90.5
0.072
7
90.8
0.055
84.9
0.059
14
92.2
0.053
83.6
0.096
30
92.2
0.021
97.0
0.082


## 3.1. LOD/LOQ Determination

As previously described, eight standards (in duplicate) were analyzed using GC-NPD: diacetyl (2.65 to 662.5 ng/mL), 2,3-pentanedione (10 to 100.7 ng/mL), and 2,3-hexanedione (5 to 201.6 ng/mL). The instrumental LOD and LOQ were determined using calibration curves (diacetyl – slope = 904.66, intercept = 15.74, andR
2
=
0.9216; 2,3-pentanedione – slope = 533.19, intercept = 29.8, and R
2
=
0.7918; 2,3-hexanedione – slope = 426.30, intercept = 2.5, and R
2
=
0.9796; and 2,3-heptanedione – slope = 113.28, intercept = 28.95, and R
2
=
0.9716). Results are listed in Table 3.Table 3
Limit of detection (LOD) and Limit of Quantitation (LOQ) for alpha-dicarbonyl compounds.
Analyte
LOD14
LOQ14
Diacetyl
7 ng/mL
23 ng/mL
2,3-Pentanedione
17 ng/mL
58 ng/mL
2,3-Hexanedione
5 ng/mL
15 ng/mL


## 3.2. Recovery Study (Untreated Silica Gel Sorbent Tubes)

On the basis of the initial recovery results achieved when diacetyl was spiked directly on untreated silica gel tubes and desorbed in a solution of 1 mg/mL ofo-PDA, a full scale recovery study was evaluated. Desorption efficiency recoveries for 2,3-dimethylquinoxaline (diacetyl derivative) ranged from 56.3% (0.0955 μg) to 104.3% (478 μg) with an average Relative Standard Deviation (RSD) of 0.039 are listed in Table 4.Table 4
Diacetyl recovery after extraction of spiked sorbent witho-PDA solution.
Spike level (μg)
Average recovery (%)
RSD
0.096
56.3
0.094
9.55
96.4
0.019
95.5
93.0
0.032
239.0
80.1
0.017
478.0
104.3
0.033


## 3.3. Recovery Study (o-PDA-Treated Silica Gel Sorbent Tubes)

The next phase in the method development process for the derivatization of diacetyl, 2,3-pentanedione, and 2,3-hexanedione was to determine the feasibility of collecting and derivatizing the analytes “on-tube” usingo-PDA-coated silica gel sorbent tubes. The initial glass wool plugs were spiked with diacetyl. Ambient air, generated by a Miller-Nelson atmospheric generator, was drawn through the tubes at 0.05 L/min. Desorption efficiencies for the analytes’ derivatization witho-PDA are depicted in Table 5.Table 5
Recovery results for the on-tube derivatization of diacetyl, 2,3-pentanedione, and 2,3-hexanedione.
Analyte
Spike level (μg)
Average recovery (%)
RSD
Diacetyl
0.096
87.0
0.106
9.55
99.6
0.037
95.5
91.4
0.099
239.0
104.0
0.080
478.0
103.0
0.077
2,3-Pentanedione
0.96∗
—∗
—∗
9.59
106.5
0.045
95.9
96.1
0.043
240.0
92.3
0.064
480.0
87.3
0.041
2,3-Hexanedione
0.117
69.7
0.212
11.7
73.5
0.032
117.0
69.6
0.032
292.0
66.8
0.040
584.0
62.5
0.028
∗Data unavailable due to sample loss during analytical preparation.


## 3.4. Low-Level Recovery Studies

As noted earlier in the Methods, after the successful recovery study at levels above 1μg, a low-level recovery study was initiated. The recoveries for diacetyl ranged from 87.2% to 100.7% with an average RSD of 0.073; for 2,3-pentanedione ranged from 94.8% to 120.1% with an average RSD of 0.068; for 2,3-hexanedione ranged from 105.1% to 117.3% with an average RSD of 0.058; and for 2,3-heptanedione ranged from 83.4% to 90.6% with an average RSD of 0.071. Results are listed in Table 6.Table 6
Low level recovery study for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione.
Analyte
Spike level (μg)
Recovery (%)
RSD
Diacetyl
0.099
87.2
0.139
0.591
96.7
0.046
0.985
100.7
0.036
2,3-Pentanedione
0.096
120.1
0.112
0.575
94.8
0.051
0.959
108.8
0.038
2,3-Hexanedione
0.084
105.1
0.043
0.504
117.3
0.056
0.840
105.1
0.043
2,3-Heptanedione
0.092
83.4
0.072
0.552
90.5
0.071
0.920
90.6
0.065


## 3.5. Studies of the Effect of Humidity on Recovery

Due to the negative effects that humidity has on diacetyl recovery discovered during some of the more recent field sampling surveys conducted at food and flavoring sites using NIOSH method 2557 [5], the next progression in our method development effort was to evaluate the effect on sample collection of various levels of humidity when using theo-PDA-coated silica gel sorbent tubes. Spiking levels ranged from approximately 0.1 μg to 500 μg. After sample collection for a period of 240 minutes and a total volume of 12 L, recoveries were determined for each derivatized analyte collected at relative humidities of 20%, 50%, and 80% (actual measured humidity). The results are listed in Table 7.Table 7
Effects of varying humidity levels on analyte recovery.
Analyte
Level (μg)
Relative humidity (%)
Mean recovery
RSD
Diacetyl
0.118
16.7
86.4
0.130
0.118
58.0
101.0
0.146
0.118
80.1
72.5
0.123
0.640
22.6
97.7
0.040
0.640
58.0
111.0
0.098
0.640
80.4
79.3
0.076
0.938
20.7
92.4
0.032
0.938
58.0
115.0
0.121
0.938
78.5
97.8
0.110
9.85
17.9
107.6
0.035
9.85
52.0
100.1
0.076
9.85
79.6
99.6
0.028
98.5
17.9
111.0
0.019
98.5
52.0
100.1
0.076
98.5
49.6
99.6
0.028
246.25
17.9
105.0
0.074
246.25
51.1
101.2
0.087
246.25
80.6
88.9
0.042
492.5
17.9
102.7
0.049
492.5
50.0
108.9
0.037
492.5
81.0
99.5
0.022
2,3-Pentanedione
0.115
16.7
63.9
0.133
0.115
58.0
98.7
0.112
0.115
80.1
73.9
0.123
0.622
22.6
88.4
0.031
0.622
58.0
85.6
0.080
0.622
80.4
76.7
0.074
0.909
20.7
84.9
0.026
0.909
58.0
102.0
0.142
0.909
78.5
98.8
0.137
9.59
21.1
94.8
0.072
9.59
51.3
85.8
0.071
9.59
80.8
64.1
0.107
95.9
21.1
102.7
0.085
95.9
51.4
100.0
0.102
95.9
79.7
46.2
0.060
239.75
21.1
104.9
0.051
239.75
50.7
99.1
0.119
239.75
80.6
73.9
0.110
479.5
21.1
90.2
0.084
479.5
50.4
87.1
0.125
479.5
79.9
80.3
0.112
2,3-Hexanedione
0.101
16.7
120.0
0.195
0.101
58.0
114.3
0.085
0.101
80.1
49.9
0.062
0.546
22.6
108.0
0.038
0.546
58.0
109.0
0.060
0.546
80.4
82.4
0.064
0.799
20.7
103.8
0.034
0.799
58.0
124.0
0.206
0.799
78.5
90.8
0.107
11.68
21.1
67.8
0.055
11.68
51.3
60.4
0.056
11.68
80.8
45.4
0.078
116.75
21.1
66.4
0.051
116.75
51.4
68.6
0.060
116.75
79.7
31.1
0.039
291.88
21.1
66.8
0.028
291.88
50.7
72.1
0.107
291.88
80.6
40.4
0.080
583.75
21.1
59.6
0.057
583.75
50.4
52.5
0.079
583.75
79.9
53.7
0.088
2,3-Heptanedione
0.110
16.7
90.3
0.180
0.110
58.0
69.8
0.142
0.110
80.1
75.9
0.062
0.598
22.6
81.0
0.034
0.598
58.0
88.7
0.066
0.598
80.4
78.4
0.061
0.874
20.7
75.3
0.067
0.874
58.0
108.0
0.195
0.874
78.5
107.0
0.111
9.20
21.5
84.9
0.099
9.20
47.3
84.5
0.053
9.20
80.3
81.9
0.077
92.0
21.5
103.1
0.044
92.0
52.7
92.3
0.030
92.0
83.8
90.2
0.031


## 3.6. Capacity Studies

In the initial collection capacity study, diacetyl, 2,3-pentanedione, and 2,3-hexanedione were sampled on theo-PDA-coated silica gel tubes at two levels (1 μg and 500 μg) for total air collection capacities ranging from 3 L (60 min) to 24 L (480 min). Sampling was conducted at relative humidities of 20% and 80% and the results are depicted in Tables 8 and 9.Table 8
Determination of air sampling capacity ofo-PDA-treated silica gel tubes at 20% relative humidity at 2 concentration levels.
Analyte
Level (μg)
Sampling volume (L)
Mean recovery (%)
RSD
Diacetyl
0.985
3
85.5
0.016
0.985
6
99.9
0.059
0.985
12
91.1
0.058
0.985
18
84.0
0.020
0.985
24
102.9
0.131
490.0
3
91.1
0.038
490.0
6
104.6
0.052
490.0
12
99.0
0.014
490.0
18
91.8
0.019
490.0
24
100.4
0.058
2,3-Pentanedione
0.959
3
105.2
0.056
0.959
6
103.3
0.085
0.959
12
105.8
0.053
0.959
18
85.8
0.016
0.959
24
106.7
0.098
480.0
3
96.3
0.038
480.0
6
97.0
0.045
480.0
12
110.6
0.028
480.0
18
116.6
0.077
480.0
24
102.3
0.039
2,3-Hexanedione
0.841
3
72.7
0.087
0.841
6
91.8
0.077
0.841
12
110.8
0.109
0.841
18
85.0
0.016
0.841
24
92.6
0.114
420.0
3
89.6
0.036
420.0
6
86.4
0.089
420.0
12
90.8
0.059
420.0
18
110.0
0.032
420.0
24
108.5
0.103Table 9
Determination of air sampling capacity ofo-PDA-treated silica gel tubes at 80% relative humidity at 2 concentration levels.
Analyte
Level (μg)
Sampling volume (L)
Mean recovery (%)
RSD
Diacetyl
0.985
3
78.1
0.016
0.985
6
82.1
0.058
0.985
12
95.2
0.097
0.985
18
82.4
0.034
0.985
24
103.6
0.028
490.0
3
97.4
0.047
490.0
6
88.5
0.074
490.0
12
112.7
0.034
490.0
18
100.6
0.018
490.0
24
93.7
0.065
2,3-Pentanedione
0.959
3
100.1
0.030
0.959
6
76.1
0.051
0.959
12
101.2
0.068
0.959
18
97.8
0.048
0.959
24
81.1
0.139
480.0
3
97.8
0.055
480.0
6
92.3
0.085
480.0
12
99.2
0.058
480.0
18
87.1
0.010
480.0
24
85.2
0.071
2,3-Hexanedione
0.841
3
100.7
0.018
0.841
6
69.5
0.077
0.841
12
98.3
0.056
0.841
18
101.4
0.027
0.841
24
80.4
0.141
420.0
3
95.5
0.049
420.0
6
92.5
0.086
420.0
12
87.7
0.053
420.0
18
94.7
0.048
420.0
24
87.6
0.173In an effort to evaluate the effect of an increased sampling rate (200 cc/min) and maximize sampling volumes collected, a more in-depth capacity study was conducted. In this study, 2,3-heptanedione was added as an analyte due to its continued presence as a minor component (contaminant) in alpha-dicarbonyl based flavoring compounds. A more detailed depiction of the recovery data for each analyte is presented in Table10 (20% RH), Table 11 (50% RH), and Table 12 (80% RH). Mean recovery (%) was calculated based on the average recovery of 3 samples evaluated at each volume sampled.Table 10
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 20% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.493
96
480
99.8
0.035
0.493
144
720
110.0
0.040
0.493
216
960
104.0
0.054
0.493
288
1440
108.0
0.043
98.5
96
480
98.3
0.010
98.5
144
720
104.0
0.023
98.5
216
960
100.0
0.038
98.5
288
1440
95.5
0.073
2,3-Pentanedione
0.479
96
480
92.1
0.015
0.479
144
720
83.0
0.046
0.479
216
960
101.0
0.088
0.479
288
1440
109.0
0.181
95.7
96
480
97.8
0.038
95.7
144
720
99.4
0.021
95.7
216
960
103.0
0.156
95.7
288
1440
95.8
0.046
2,3-Hexanedione
0.420
96
480
97.1
0.021
0.420
144
720
102.0
0.036
0.420
216
960
109.0
0.016
0.420
288
1440
106.0
0.061
84.1
96
480
87.9
0.036
84.1
144
720
101.0
0.021
84.1
216
960
99.4
0.032
84.1
288
1440
84.6
0.047
2,3-Heptanedione
0.460
96
480
104.0
0.027
0.460
144
720
101.0
0.065
0.460
216
960
106.0
0.025
0.460
288
1440
108.0
0.052
92.0
96
480
93.9
0.082
92.0
144
720
98.6
0.029
92.0
216
960
104.0
0.024
92.0
288
1440
93.5
0.052Table 11
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 50% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.493
96
480
102.0
0.015
0.493
144
720
92.6
0.024
0.493
216
960
102.0
0.018
0.493
288
1440
87.0
0.026
98.5
96
480
98.8
0.027
98.5
144
720
106.0
0.016
98.5
216
960
101.8
0.020
98.5
288
1440
101.0
0.028
2,3-Pentanedione
0.479
96
480
92.7
0.010
0.479
144
720
77.8
0.054
0.479
216
960
98.5
0.158
0.479
288
1440
88.5
0.009
95.7
96
480
97.8
0.030
95.7
144
720
100.0
0.019
95.7
216
960
110.0
0.024
95.7
288
1440
97.8
0.026
2,3-Hexanedione
0.420
96
480
93.7
0.024
0.420
144
720
86.2
0.021
0.420
216
960
87.9
0.020
0.420
288
1440
71.4
0.018
84.1
96
480
88.0
0.023
84.1
144
720
102.0
0.023
84.1
216
960
103.0
0.026
84.1
288
1440
89.3
0.018
2,3-Heptanedione
0.460
96
480
103.0
0.019
0.460
144
720
89.3
0.026
0.460
216
960
97.8
0.031
0.460
288
1440
98.5
0.041
92.0
96
480
94.1
0.020
92.0
144
720
99.8
0.017
92.0
216
960
105.0
0.011
92.0
288
1440
98.6
0.019Table 12
Determination of extended air sampling capacity ofo-PDA-treated silica gel tubes at 80% relative humidity at 2 concentration levels using increased sampling rate (200 cc/min).
Analyte
Level (μg)
Sampling volume (L)
Time (min)
Mean recovery (%)
RSD
Diacetyl
0.985
96
480
99.7
0.017
0.985
144
720
101.0
0.110
0.985
216
960
93.7
0.055
0.985
288
1440
84.1
0.114
98.5
96
480
90.5
0.072
98.5
144
720
98.8
0.036
98.5
216
960
93.5
0.057
98.5
288
1440
89.4
0.037
2,3-Pentanedione
0.957
96
480
72.9
0.081
0.957
144
720
112.0
0.055
0.957
216
960
105.0
0.114
0.957
288
1440
99.5
0.054
95.7
96
480
86.0
0.055
95.7
144
720
93.8
0.038
95.7
216
960
55.2
0.054
95.7
288
1440
48.8
0.035
2,3-Hexanedione
0.841
96
480
119.0
0.138
0.841
144
720
122.0
0.062
0.841
216
960
105.0
0.114
0.841
288
1440
44.8
0.031
84.1
96
480
86.2
0.060
84.1
144
720
93.5
0.039
84.1
216
960
60.4
0.053
84.1
288
1440
54.6
0.015
2,3-Heptanedione
0.920
96
480
52.0
0.021
0.920
144
720
104.0
0.089
0.920
216
960
76.0
0.154
0.920
288
1440
57.2
0.055
92.0
96
480
85.5
0.058
92.0
144
720
93.7
0.036
92.0
216
960
57.8
0.050
92.0
288
1440
53.7
0.018


## 3.7. Storage Stability Recoveries

Evaluation of the ambient and refrigerated storage stability recovery results for diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione indicates that the derivatized analytes were stable for up to 30 days at the 0.6μg spiking levels. The average storage stability results evaluated for each analyte at 1, 7, 14, and 30 days are reported in Table 13.Table 13
Storage stability conducted under ambient and refrigerated storage conditions.
Day
Avg. recovery (ambient, %)
RSD
Avg. recovery (refrigerated, %)
RSD
Diacetyl
1
96.1
0.068
96.7
0.046
7
93.4
0.048
89.8
0.039
14
98.6
0.017
106.0
0.065
30
103.0
0.039
98.6
0.047
2,3-Pentanedione
1
98.0
0.049
94.8
0.051
7
102.0
0.045
100.0
0.025
14
105.0
0.091
97.1
0.068
30
108.0
0.065
96.8
0.073
2,3-Hexanedione
1
109.8
0.115
117.0
0.056
7
93.9
0.041
110.0
0.054
14
87.0
0.068
99.0
0.040
30
81.4
0.026
99.6
0.053
2,3-Heptanedione
1
89.3
0.109
90.5
0.072
7
90.8
0.055
84.9
0.059
14
92.2
0.053
83.6
0.096
30
92.2
0.021
97.0
0.082


## 4. Discussion and Conclusions

A method for alpha-dicarbonyl flavoring compounds has been developed using derivatization witho-PDA. This method has several advantages when compared to other methods [3, 6, 7] for alpha-dicarbonyl flavoring compounds, such as improved sensitivity (instrumental LODs of 5–17 ng/sample), use of a single sampling tube amenable to on-tube derivatization of the analytes of interest, longer sampling times, variable sampling rates, and greater sampling capacity (up to 288 L with low-to-moderate humidity). Chromatographic separation of the alpha-dicarbonyl derivatives was good and the overall recovery of the analytes of interest down to the 0.1 μg level was acceptable.Diacetyl recoveries on untreated silica gel tubes following by desorption in ethanol containing theo-PDA derivatizing agent were acceptable at all spiking levels except the lowest (0.096 ng).Recoveries of diacetyl and 2,3-pentanedione from the silica gel tubes coated witho-phenylenediamine were very good while the recoveries for 2,3-hexanedione were approximately 20% lower. Lower recoveries of 2,3-hexanedione and 2,3-heptanedione may be the result of the increasing hydrocarbon nature of these compounds and/or the fact that they possibly require an increased derivatization period. In addition, when larger amounts of the analyte were evaluated, some lower recoveries were found. This may be the result of incomplete derivatization and the need for a greater concentration of the derivatizing reagent on the sorbent media at these higher levels and/or the fact that the higher concentrations evaluated may exceed the sampling capacity of the sorbent tubes. The recoveries for 2,3-hexanedione and 2,3-heptanedione are lower than what is normally considered acceptable [13]. While this method was developed for diacetyl and 2,3-pentanedione measurement to address humidity issues with existing methods, it can be used to determine the presence of larger chain alpha-dicarbonyl compounds that may be present as by-products.Sample collection for diacetyl was unaffected by humidity ranging from 20% to 80%. For the other flavoring agents tested, high humidity reduced the recovery. While additional research studies are ongoing, it can be reasonably concluded that this method has achieved significant advancements in the sampling and quantitation of alpha-dicarbonyl flavoring compounds. Overall, recoveries were good for diacetyl when sampled in conditions of 80% humidity.The resulting recovery for 2,3-pentanedione at 95.9μg (46.2%) is abnormally low when compared to all other results and is most likely an aberration when compared to the results listed in Table 9 for 2,3-pentanedione. Recoveries for both 2,3-hexanedione and 2,3-heptanedione were lower than expected for those samples collected at 80% humidity in the extended air sampling capacity studies. These analytes are present as by-products or contaminants with either diacetyl or 2,3-pentanedione. This method can be used to detect these contaminants where other methods cannot.In laboratory capacity studies, where diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione were collected at 20% relative humidity and with a sampling rate of 200 cc/min, all three analytes exhibited acceptable recoveries (>80%) with little variation in the mean recoveries when sampling for a total volume of 288 L. At 50% relative humidity, diacetyl, 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione exhibited acceptable recoveries. Results for the samples collected for the 100μg level showed that 2,3-pentanedione, 2,3-hexanedione, and 2,3-heptanedione had significant decreases in recovery at a collection volume of 216 L. These results would seem to suggest that the maximum sampling volume for these analytes, when collected at a higher sampling rate (200 cc/min), would be between 144 and 216 L.The average 30-day storage stability recovery results, almost quantitative in nature, are extremely good and acceptable for both the ambient and refrigerated samples of the derivatized diacetyl and 2,3-pentanedione, suggesting that temperature does not have either a positive or a negative effect on the derivatization and storage of the diacetyl samples. Since both the ambient and refrigerated samples had quantitative recoveries (>95%) and RSD values less than 1% [13], there is no difference between the two methods of storage.Evaluation of both ambient and refrigerated storage stability recovery results for 2,3-hexanedione indicates that the derivatized analyte was stable for up to 30 days at the 0.6μg spiking levels. Analysis of the results indicates that there is improved storage stability (18% increase in average recovery) when the samples are refrigerated. This is especially true for the samples analyzed after 14 and 30 days. Evaluation of both ambient and refrigerated storage stability recovery results for 2,3-heptanedione indicates that the derivatized analyte was stable for 30 days. Comparison of the averaged recoveries for both the ambient and refrigerated samples of the derivatized 2,3-heptanedione revealed no differences based on storage temperature. Storage stability studies indicated that the compounds of interest, especially diacetyl and 2,3-pentanedione, as their quinoxaline derivatives, are stable at both ambient and refrigerated temperatures for 30 days. Separation of other alpha-dicarbonyls such as 2,3-hexanedione and 2,3-heptanedione can be achieved with this method and provide semiquantitative results. Overall, this method may be another useful tool for the evaluation and monitoring of workers exposed to airborne alpha-dicarbonyl food and flavoring compounds. Additional laboratory and field studies using the method are necessary to obtain full validation and publication in the NIOSH Manual of Analytical Methods (NMAM).In summary, to date, all results suggest that this method provides the sensitivity needed for nanogram level sampling for alpha-dicarbonyl food and flavoring compounds (diacetyl and 2,3-pentanedione). Additionally, the method allows collection over a wide mass range and at relative humidities ranging from 20% to 80%, with acceptable recoveries achieved up to sampling volumes of 144 L and 288 L for 2,3-pentanedione and diacetyl, respectively. The sampling and analytical methodology has been unaffected by breakthrough when sampling at high flow rates (200 cc/minute) and high sample collection volumes (144 L), eliminating the need for a second sorbent tube in series with the backup section of the single sorbent tube collecting any sample that breaks through. Additionally, the on-tube derivatization eliminates humidity-related breakthrough of the alpha-dicarbonyl flavoring compounds by forming the stable quinoxaline derivatives.



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*Source: 9059678-2016-05-10.xml*