Determination of 2-Methylimidazole, 4-Methylimidazole, and 2-Acetyl

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Determination of 2-Methylimidazole, 4-Methylimidazole and 2-Acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in Liquorice using High Performance Liquid ChromatographyTandem Mass Spectrometry Stable-Isotope Dilution Analysis Marion Raters, Paul Wilhelm Elsinghorst, Stephanie Goetze, and Reinhard Matissek J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01493 • Publication Date (Web): 13 Jun 2015 Downloaded from http://pubs.acs.org on June 18, 2015

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Journal of Agricultural and Food Chemistry

Determination

of

2-Methylimidazole,

4-Methylimidazole

and

2-Acetyl-4-(1,2,3,4-

tetrahydroxybutyl)imidazole in Liquorice using High Performance Liquid ChromatographyTandem Mass Spectrometry Stable-Isotope Dilution Analysis Marion Raters*‡, Paul Elsinghorst†, Stephanie Goetze‡, Anna Dingel‡, and Reinhard Matissek‡ ‡

Food Chemistry Institute of the Association of the German Confectionery Industry, Adamsstraße

52-54, D-51063 Köln, Germany †

ELFI Analytik GbR, Massenhausener Strasse 18a, D-85375 Neufahrn, Germany

*

Corresponding author: Dr. Marion Raters, Food Chemistry Institute of the Association of the

German Confectionery Industry, Adamsstraße 52-54, D-51063 Köln, Germany; Phone: +49-221623061, Fax: +49-221-610477, e-mail: [email protected]

1 ACS Paragon Plus Environment


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1

Abstract

2

A quick and selective analytical method was developed for the simultaneous quantitation of 2-

3

methylimidazole, 4-methylimidazole, and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole, which

4

are known to be formed by Maillard reactions. The methodology reported here employs stable-

5

isotope dilution analysis (SIDA) using 4-methylimidazole-d6 and [13C6]-2-acetyl-4-(1,2,3,4-

6

tetrahydroxybutyl)imidazole as internal standards. It was successfully applied in a model assay to

7

show that the addition of ammonium chloride during manufacture of liquorice promotes imidazole

8

formation depending on the added amount of ammonium chloride without the well-known impact

9

of present caramel food colorings. Furthermore a monitoring assay of 29 caramel coloring-free

10

liquorice

products

showed

that

both,

4-methylimidazole

and

2-acetyl-4-(1,2,3,4-

11

tetrahydroxybutyl)imidazole, are endogenously generated in detectable quantities. None of the

12

samples showed 2-methylimidazole levels above the limit of detection, 50 µg/kg.

13

14

Keywords: methylimidazole, 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole, liquorice, LC–

15

MS/MS, SIDA

16

17

Introduction

18

2-Methylimidazole (1), 4-methylimidazole (2), and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole

19

(3) are nitrogen-containing, heterocyclic, aromatic compounds (Figure 1), which appear as process

20

contaminants during the manufacture of caramel food colorings. Their formation results from

21

Maillard reactions (non-enzymatic browning) between reducing sugars and amino compounds as

22

shown for 2 in Figure 1.1 Whether these imidazole derivatives might also be generated when


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23

ammonium chloride is used for the flavoring of liquorice products is hitherto unknown and was one

24

subject of the present study.

25

In 2011, 1 and 2 were classified as “possibly carcinogenic to humans” (2B) by the

26

International Agency for Research on Cancer (IARC) and 3 has been reported to exert

27

immunosuppressive effects.2-4 Subsequently, Commission Regulation (EU) No 231/2012 came into

28

effect on December 1, 2012 including maximum limits for 2 and 3 in ammonia caramel food

29

colorings (E150c) (2: 200 mg/kg, 3: 10 mg/kg; on an equivalent color basis, i.e., a color intensity of

30

0.1 absorbance units) as well as in sulfite ammonia caramel food colorings (E150d) (2: 250 mg/kg;

31

on an equivalent color basis),5 which had previously been regulated in Commission

32

Directive 2008/128/EC.6 Furthermore, as Commission Implementing Regulation (EU) No 872/2012

33

entered into force on April 22, 2013, no maximum limit (20 g/kg, 2%) for the use of ammonium

34

chloride in liquorice products applies any longer and has been replaced by the quantum satis

35

principle.7 Hence the question arose, whether the above-mentioned imidazoles could also be formed

36

during liquorice manufacture with ammonium chloride as a source of nitrogen, even though the

37

high temperatures of 120-160 °C applied for production of caramel food colorings would not be

38

reached. This question was to be addressed in this study.

39

Analysis of the considered imidazoles is typically carried out by liquid chromatography-

40

tandem mass spectrometry (LC–MS/MS) as their analysis by gas chromatography usually involves

41

laborious derivatization reactions.8,9 The available literature describes various methods for the

42

determination of 1-3 in different matrices, however, only a few published methods exist for their

43

simultaneous quantitation.10,11 For example, Wang and Schnute described a UHPLC–MS/MS

44

methodology using 4-methylimidazole-d3 as an internal standard for their simultaneous

45

determination in beverages using basic eluents under isocratic conditions on a C30 separation

46

phase.10 Schlee et al. published a method for the quantitation of 1-3 in caramel food colorings and

47

Cola beverages by LC–MS/MS with gradient elution under basic condition on a common C18

48

column without the use of an internal standard.11 3 ACS Paragon Plus Environment


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Here we present an analytical method for the simultaneous and selective determination of

50

the imidazoles 1-3 in liquorice and other foodstuffs by high performance liquid chromatography-

51

tandem mass spectrometry applying stable-isotope dilution analysis (SIDA). Chromatographic

52

separation was achieved on a common C18 reversed-phase column within three minutes. Detection

53

was carried out with the MS/MS system operating in multiple reaction monitoring (MRM) mode.

54

Materials and Methods

55

Chemicals and Reagents. 1 and 2 were obtained from Sigma-Aldrich (Taufkirchen, Germany), 3

56

and [13C6]-2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (5, see Figure 1) were from ELFI

57

Analytik GbR (Neufahrn, Germany), and 4-methylimidazole-d6 (4, see Figure 1) was from Dr.

58

Ehrenstorfer (Augsburg, Germany). HPLC-grade methanol was purchased from Bernd Kraft

59

(Duisburg, Germany), while formic acid, ammonia, potassium hexacyanoferrate(II) trihydrate and

60

zinc sulfate heptahydrate of analytical grade were from Merck (Darmstadt, Germany). Liquorice

61

samples (ammonium chloride content ≤ 2%) were purchased in local retail stores. Liquorice

62

products for the model assay were provided by the testing facility of a major German liquorice

63

manufacturer.

64

Stock Solutions. Stock solutions of 1, 2, and 4 were prepared by dissolving a suitable amount of

65

the respective chemical in a mixture of methanol and purified water (MeOH/H2O, 50/50 v/v) to

66

obtain a concentration of 1 mg/mL. Stock solutions of 3 and 5 were prepared by dissolving a

67

suitable amount of the respective substance in 0.1M hydrochloric acid. Calibration levels of all

68

analytes and internal standards were prepared by dilution of the respective stock solution with

69

MeOH/H2O (50/50 v/v) to obtain a respective concentration of 5 µg/mL. A calibration standard

70

solution was prepared by mixing the standard solutions of 1, 2, 3, 4, and 5 in MeOH/H2O

71

(50/50 v/v) and contained 5 ng/mL of 1, 2, and 4 as well as 10 ng/mL of 3 and 5. This solution was

72

further diluted with purified water (1:10). Internal standard solutions of 4 and 5 were prepared by


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73

appropriate dilution of the stock solutions with MeOH/H2O (50/50 v/v) to a final concentration of

74

100 µg/mL.

75

Sample Preparation. All samples were finely ground and stored at room temperature prior to

76

analysis. For equilibration, each sample (4 g) was placed in a 100 mL iodine determination flask,

77

spiked with 4 (5 ng/mL, 200 µL) and 5 (10 ng/mL, 150 µL) and incubated at room temperature for

78

30 min. For extraction of the imidazoles methanol/water (50/50 v/v, 50 mL) was added and the

79

mixture was stirred 30 min, followed by another volume of methanol/water (50/50 v/v, 50 mL) and

80

subsequent sonication for 15 min at room temperature. Removal of proteins was achieved following

81

the procedure of Carrez (150 g potassium hexacyanoferrate(II) trihydrate/L, 300 g zinc sulfate

82

heptahydrate/L; 500 µL each). Finally, after filtration through a folded filter (Whatman, Germany)

83

the obtained filtrates were diluted (for analysis of 2 and 3: purified water 1:10; for analysis of 1:

84

MeOH/H2O 1/20 v/v 1:20) into HPLC vials.

85

LC–MS/MS Conditions. The LC–MS/MS system consisted of a 1260 Infinity HPLC from Agilent

86

(Waldbronn, Germany) coupled to a TripleQuad 4500 tandem mass spectrometer from ABSCIEX

87

(Darmstadt, Germany) through an electrospray ionisation (ESI) interface in positive ionisation

88

mode (spray voltage: 3.0 kV, vaporizer temperature: 450 °C, curtain gas: 25 psi N2, collision energy

89

25 V; Turbo V, ABSCIEX). Chromatographic separation was achieved on a Poroshell 120 EC-C18

90

(4.6 × 50 mm, 2.7 µm particle size, Agilent) under isocratic conditions (A/B, 50/50 v/v); A: of 0.1%

91

formic acid/methanol (99.5/0.5 v/v), B 0.05% ammonia solution/methanol (90/10 v/v). The injection

92

volume was 20 µL and the flow rate was set to 0.45 mL/min. The following MRM transitions were

93

used for analyte detection: 1 (m/z 83.0 → 42.0 (target), m/z 83.0 → 56.0 (qualifier)), 2 (m/z

94

83.0 → 56.0 (target), m/z 83.0 → 42.0 (qualifier)), 4 (m/z 89.0 → 62.0 (target), m/z 89.0 → 48.0

95

(qualifier)), 3 (m/z 231.0 → 195.0 (target), m/z 231.0 → 153.0 (qualifier)), 5 (m/z 237.0 → 201.0

96

(target), m/z 237.0 → 159.0 (qualifier)).



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97

Method Validation. Linearity was assessed by spiking a blank sample (liquorice-free candy) at 10

98

different levels (0.005, 0.01, 0.025, 0.05, 0.10, 0.50, 1.00, 1.50, 2.50, and 5.00 mg/kg of 1-3). LODs

99

and LOQs of 1-3 were derived according to DIN 32645 by tenfold analysis of a blank sample

100

(direct method according to 5.3 of DIN 32645).15 Total recovery was evaluated by spiking a blank

101

sample to a final content of 1 mg/kg 1-3 followed by sixfold analysis. For the determination of

102

intra-day accuracy and inter-day precision a liquorice sample obtained from a local retail store was

103

used.

104

Results and Discussion

105

Method Development. One objective of this study was to develop and validate a method for the

106

sensitive and simultaneous quantitation of the imidazoles 1, 2, and 3. Quantitative determination

107

was carried out using commercially available 4 and 5 as internal standards (Figure 1). To achieve

108

baseline separation of the 1 and 2 regioisomers, various parameters like eluent composition, flow

109

rate, temperature and column material were evaluated. Separation of the analytes was tested on a

110

C18 (Poroshell 120 EC-C18, Agilent) or a C30 (Acclaim C30, Thermo Scientific, Dreieich,

111

Germany) reversed-phase as well as on a porous graphitic carbon phase (Hypercarb, Thermo

112

Scientific) while using a two-component eluent composed of A (0.05% aqueous ammonia solution,

113

100%, 0-2 min) and B (methanol, 100%, 2-3 min) at a flow of 0.45 mL/min. In addition, an

114

isocratic eluent made up from acidic and alkaline components (0.1% formic acid/methanol

115

99.5/0.5 v/v; 0.05% ammonia solution/methanol, 90/10 v/v) was evaluated, which, when used in

116

combination with the Poroshell 120 EC-C18 column, provided an excellent baseline separation of 1,

117

2, and 3 (Figure 2). Hence the Poroshell 120 EC-C18 column and an eluent composed of acidic and

118

basic components were used for further method development.

119

When analyzing complex liquorice matrices, ion suppression was observed for 1 when

120

compared to the aqueous samples used for chromatography development. To remove the interfering

121

matrix components a common Carrez clarification was introduced and several dilution steps (1:10,


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122

1:100, 1:200 v/v) of the sample solution injected into the LC–MS/MS system were evaluated to

123

eliminate any remaining matrix effects. A recovery for 1 of 86% with acceptable sensitivity was

124

finally achieved at a dilution of 1:200.

125

Although baseline-separated, the 1 and 2 regioisomers can also be distinguished from

126

differences in their fragmentation behavior. While 1 shows higher relative intensities at mass

127

transition m/z 83 → 42, 2 shows higher intensity at mass transition m/z 83 → 56 (Figure 2).

128

Figure 3 shows a typical chromatogram of a liquorice sample recorded using the optimized LC–

129

MS/MS conditions described above.

130

Method Validation. Following the guidelines laid out by Kromidas,14 method validation included

131

the following parameters: intra-day accuracy, inter-day precision, recovery, linearity, limit of

132

detection (LOD), and limit of quantitation (LOQ). Validation data are summarized in Table 1.

133

Limits of detection and quantitation were estimated according to DIN 32645 and were

134

0.05 mg/kg for 1, 0.02 mg/kg for 2, and 0.01 mg/kg for 3 (LOD) as well as 0.03-0.16 mg/kg for all

135

analytes (LOQ).15 Linearity was evaluated by plotting the concentration of each analyte against the

136

respective analyte/internal standard ratio (2 was calibrated using 4 as the internal standard).

137

Linearity in the range of LOD to 5 mg/kg of liquorice was shown for all three analytes by a

138

coefficient of determination ≥ 0.999. Intra-day accuracies and inter-day precisions were calculated

139

from twelve separate measurements and showed standard deviations ≤ 10.2% (Table 1). The

140

developed methodology provided efficient recovery rates, which are in accordance with available

141

literature data: 1 (86%), 2 (99%), 3 (100%).10,11

142

Endogenous Formation. To check for endogenous formation of the imidazoles 1, 2, and 3 in

143

liquorice in the presence of ammonium chloride a model assay was conducted examining several

144

coloring-free liquorice products (n = 20, five levels of ammonium chloride in duplicate: 1, 2, 4, 6,

145

and 8%). Depending on their manufacture samples were further grouped into in-house types A, B,

146

and C, where ammonium chloride had either been added to the cooking mass before (type A) or

147

after (type B) the cooking process, while type C (here not under consideration) was simply dredged 7 ACS Paragon Plus Environment


Page 8 of 17

148

in solid ammonium chloride (see also Supplementary Information, Figure S1). All model assays

149

were conducted under typical industrial conditions at a testing facility of a major German liquorice

150

manufacturer and samples were analyzed in our laboratory.

151

The results show a linear correlation (r2 = 0.937) between the levels of endogenously

152

formed 2 and the amount of added and concomitantly heated ammonium chloride when the

153

ammonium chloride was added to the cooking mass before the cooking process (type A). The

154

determined absolute levels of 2 (relative to the dry mass) were in the range of 0.02 (LOD) to

155

1.5 mg/kg. In type B liquorice products, where ammonium chloride is added after the cooking

156

process, most of the samples showed levels of 2 below the LOQ of 0.07 mg/kg. Only those products

157

with 8% ammonium chloride showed levels at 0.09 mg/kg just above the LOQ.

158

Endogenous formation of 3 in type A liquorice products again depends on the amount of

159

added and concomitantly heated ammonium chloride. A linear correlation between the

160

endogenously generated amounts of 3 and the added ammonium chloride was derived with a

161

coefficient of determination of r2 = 0.909. The determined levels of 3 (relative to the dry mass)

162

were in the range of 0.01 (LOD) to 0.17 mg/kg. In the case of type B liquorice products all samples

163

showed levels below an LOQ of 0.03 mg/kg.

164

Monitoring. Monitoring assays were subsequently carried out to examine the endogenous

165

formation of 1-3 in 29 coloring-free liquorice products. 1 was not observed in any of the samples,

166

while levels of 2 and 3 were in the range of LOD-1.30 mg/kg (median: < LOQ) and LOD-

167

0.10 mg/kg (median: < LOQ), respectively.

168

Maximum limits for 2 and 3 have not been defined for end products but are available for the

169

caramel food colorings ammonia caramel E150c and sulphite ammonia caramel E150d (E150c:

170

200 mg/kg 2, 10 mg/kg 3; E150d: 250 mg/kg 2).5 Assuming that an amount of caramel food

171

colorings of up to 2% is usually added to certain liquorice products, the resultant carry-over

172

produces a still acceptable content level for 2 of 4 mg/kg and a content level of 0.2 mg/kg for 3.

173

However, the determined levels of 2 and 3 formed in commercial liquorice products by addition of 8 ACS Paragon Plus Environment

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ammonium chloride were well below these theoretically derived maximum levels. The liquorice

175

products reviewed in this study can thus be judged safe with respect to a possible contamination by

176

any of the imidazoles 1-3.

177

Supporting Information. A schematic overview of the manufacture of type A, B, and C liquorice

178

products. This material is available free of charge via the Internet at http://pubs.acs.org.

179



180

References

181

(1)

182 183

Moon, J. K.; Shibamoto, T. Formation of carcinogenic 4(5)-methylimidazole in Maillard reaction systems. J. Agric. Food Chem. 2011, 59, 615–618.

(2)

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC

184

Monographs on the Evaluation of Carcinogenic Risks to Humans 101 (14). 2012.

185

http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-014.pdf (accessed: Jan 20,

186

2015).

187

(3)

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC

188

Monographs on the Evaluation of Carcinogenic Risks to Humans 101 (15). 2012.

189

http://monographs.iarc.fr/ENG/Monographs/vol101/mono101-015.pdf (accessed: Jan 20,

190

2015).

191

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(4)

Houben, G. F.; van Dokkum, W.; van Loveren, H.; Penninks, A. H.; Seinen, W.; Spanhaak,

192

S.; Ockhuizen, T. Effects of Caramel Colour III on the number of blood lymphocytes: A

193

human study on Caramel Colour III immunotoxicity and a comparison of the results with data

194

from rat studies. Food Chem. Toxicol. 1992, 30, 427–430.

195

(5)

Commission Regulation (EU) No 231/2012 of 9 March 2012 laying down specifications for

196

food additives listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European

197

Parliament and of the Council Text with EEA relevance. Off. J. Eur. Communities: Legis.

198

2012, L83, 1–295.

199

(6)

200 201

Commission Directive 2008/128/EC laying down specific purity criteria concerning colours for use in foodstuffs. Off. J. Eur. Communities: Legis. 2008, L6, 20–63.

(7)

Commission Implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list

202

of flavouring substances provided for by Regulation (EC) No 2232/96 of the European

203

Parliament and of the Council, introducing it in Annex I to Regulation (EC) No 1334/2008 of

204

the European Parliament and of the Council and repealing Commission Regulation (EC) No

205

1565/2000 and Commission Decision 1999/217/EC. Off. J. Eur. Communities: Legis. 2012, 10 ACS Paragon Plus Environment

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206 207

L267, 1–161. (8)

Fernandes, J. O.; Ferreira, M. A. Gas chromatographic-mass spectrometric determination of

208

4-(5) methylimidazole in ammonia caramel colour using ion-pair extraction and derivatization

209

with isobutylchloroformate. J. Chromatogr. A. 1997, 786, 299–308.

210

(9)

Casal, S.; Fernandes, J. O.; Oliveira, M. B. P. P.;Ferreira, M. A. (2002). Gas

211

chromatographic–mass spectrometric quantification of 4-(5-)methylimidazole in roasted

212

coffee after ion-pair extraction. J. Chromatogr. A. 2002, 976, 285–291.

213

(10) Wang, J.; Schnute, W. C. Simultaneous Quantitation of 2-Acetyl-4-tetrahydroxybutyl-

214

imidazole, 2- and 4-Methylimidazoles, and 5-Hydroxymethylfurfural in Beverages by

215

Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry. J. Agric. Food

216

Chem. 2012, 60, 917–921.

217

(11) Schlee, C.; Markova, M.; Schrank, J.; Laplagne, F.; Schneider, R.; Lachenmeier, D. W.

218

Determination of 2-methylimidazole, 4-methylimidazole and 2-acetyl-4-(1,2,3,4-

219

tetrahydroxy-butyl)imidazole in caramel colours and cola using LC/MS/MS. J. Chromatogr.

220

B. 2012, 927, 223–226.

221

(12) Dingel, A.; Goetze, S.; Raters, M.; Elsinghorst, P.; Matissek, R. Quantifizierung der

222

Imidazole 4-MEI und THI in Zuckerkulör und anderen Lebensmitteln mittels SIVA-LC–

223

MS/MS. Lebensmittelchemie 2013, 67, 166.

224

(13) Elsinghorst, P. W.; Raters, M.; Dingel, A.; Fischer, J.; Matissek, R. Synthesis and Application

225

of 13 C-Labeled 2-Acetyl-4-((1 R ,2 S ,3 R )-1,2,3,4-tetrahydroxybutyl)-imidazole (THI), an

226

Immunosuppressant Observed in Caramel Food Colorings. J. Agric. Food Chem. 2013, 61,

227

7494–7499.

228

(14) Kromidas, S. Handbuch Validierung in der Analytik. Wiley-VCH, Weinheim. 2000.

229

(15) DIN 32645:2008-11. Chemical analysis - Decision limit, detection limit and determination

230

limit under repeatability conditions - Terms, methods, evaluation. Beuth Verlag GmbH,

231

Berlin. 2008. 11 ACS Paragon Plus Environment


Page 12 of 17

Figure Captions

Figure 1. Formation of 4-methylimidazole (2) and chemical structures of 2-methylimidazole (1), 2acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (3), as well as of the internal standards (IS) 4methylimidazole-d6 (4) and [13C6]-2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (5, * = 13C).

Figure 2. LC–MS/MS mass traces of the calibration standard solution at concentration levels of 0.51 ng/mL for 2-methylimidazole (1), 0.50 ng/mL for 4-methylimidazole (2), and 0.94 ng/mL for 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (3).

Figure 3. Example chromatogram of a liquorice sample: 2-methylimidazole (1) < LOD, 4methylimidazole

(2) = 1.09 mg/kg,

and

2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole

(3) = 0.12 mg/kg.


Page 13 of 17


Figure 1



Page 14 of 17

Figure 2 1

2

1

83.0 → 42.0

2 83.0 → 56.0

IS: 4

89.0 → 62.0

3

231.0 → 195.0

IS: 5 237.0 → 201.0


Page 15 of 17


Figure 3 1

2 83.0 → 42.0

2 83.0 → 56.0

IS: 4 89.0 → 62.0

3 231.0 → 195.0

IS: 5 237.0 → 201.0



Page 16 of 17

Table 1. Method validation data. 2-acetyl-4-(1,2,3,442tetrahydroxybutyl) methylimidazole methylimidazole imidazole 0.05-5.00 0.02-5.00 0.01-5.00 2 2 (r ≥ 0.999) (r ≥ 0.999) (r2 ≥ 0.999)

calibration range (mg/kg) recovery (%)

86.1

99.4

99.5

limit of detection (LOD, mg/kg)

0.05

0.02

0.01

limit of quantitation (LOQ, mg/kg)

0.16

0.07

0.03

intra-day accuracy (n = 12, %)

–

2.2

5.5

inter-day precision (n = 12, %)

–

9.3

10.2


Page 17 of 17


4-MI (mg/kg dry mass)

THI (mg/kg dry

TOC graphic

0.20 0.16 0.12 0.08 0.04 0.00 0.0

3.0

6.0

9.0

c (NH4Cl, type A) (%)

2.0 1.5 1.0 0.5 0.0 0.0

3.0

6.0

9.0

c (NH4Cl, type A) (%)


Determination of 2-Methylimidazole, 4-Methylimidazole, and 2-Acetyl

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