Validation of a sensitive high performance liquid chromatography tandem mass spectrometric method for measuring carbohydrates in aerosol samples

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Validation of a sensitive high performance liquid chromatography tandem mass spectrometric method for measuring carbohydrates in aerosol samples Wenjing Li , Mindong Chen , Xinlei Ge , Chuanxin Gu , Wentao Yu , Dongyang Nie PII: DOI: Reference:

S0021-9673(20)30128-X https://doi.org/10.1016/j.chroma.2020.460941 CHROMA 460941

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

11 November 2019 30 January 2020 1 February 2020

Please cite this article as: Wenjing Li , Mindong Chen , Xinlei Ge , Chuanxin Gu , Wentao Yu , Dongyang Nie , Validation of a sensitive high performance liquid chromatography tandem mass spectrometric method for measuring carbohydrates in aerosol samples, Journal of Chromatography A (2020), doi: https://doi.org/10.1016/j.chroma.2020.460941

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Highlights 

A sensitive method based on HPLC-MS/MS was developed to determine carbohydrates in atmosphere.



This may be the first time to attach CH3COO- to 2-Methylbutane-1,2,3,4-tretraol using HPLC-MS/MS.



There is no need to separate carbohydrates completely in SRM scan mode.



The LODs of carbohydrates may be the lowest in HPLC-MS/MS methods.

1

Validation of a sensitive high performance liquid chromatography tandem mass spectrometric method for measuring carbohydrates in aerosol samples Wenjing Li, Mindong Chen*, Xinlei Ge*, Chuanxin Gu, Wentao Yu, Dongyang Nie Collaborative Innovation Center of Atmospheric Environment and Equipment Technology; Jiangsu key laboratory of Atmospheric Environment Monitoring and Pollution Control, School of Environmental Science and Engineering, Nanjing University of information Science & Technology; Nanjing, 210044, China

Abstract Carbohydrates (such as levoglucosan) are a class of important water-soluble organic compounds in atmosphere. In this study, a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was applied to characterize carbohydrates in aerosol particles. Since carbohydrate was a kind of compound with low response in mass spectrometry, the conventional HPLC-MS/MS method was not sensitive enough to determine them. When acetate acid was added into mobile phase as buffer solution, the transition of [M+CH 3COO]-→[M-H]could be selected as the quantification ions. In the range from 1.0 μg L -1 to 20 μg mL-1, the coefficients of regression (r2) were more than 0.990, and relative standard deviations (RSD) for replicate injections were lower than 2%. The limit of detection (LOD) and quantification (LOQ) were lower than 2.5 ng L-1 and 10 ng L-1, respectively. The precision and accuracy were examined by spiked samples at three different concentrations levels (10 μg L -1, 100 μg L-1, and 500 μg L-1) in five replicates. Recovery rations ranged from 85%-115% with RSD lower than 16%. Matrix effects of different carbohydrates ranged from 62% to 120%. The most sensitive HPLC-MS/MS method was developed and validated to analyze 40 aerosol samples successfully. The carbohydrates including three sugar alcohols (threitol, arabitol and sorbitol), one monosaccharide sugar (inositol), two disaccharides (sucrose, trehalose) and one anhydrosugar (levoglucosan) and 2

one 2-methyltetrols (2-Methylbutane-1,2,3,4-tretraol) were successfully quantified. Key words: Carbohydrates, HPLC-MS/MS, sensitivity, aerosol. 1. Introduction Carbohydrates are a class of important water-soluble organic compounds in atmosphere [1,2,3,4,5,6,7,8,9,10]. Levoglucosan, largely derived from the pyrolysis of cellulose, has been commonly regarded as an important tracer of biomass burning [11,12,13,14,15]. There are innumerable sources of sugar and sugar alcohols in the atmosphere, including biological aerosols such as pollen, fungal spores, plant debris, viruses and bacteria [16]. To be specific, arabitol and mannitol, for example, are the airborne tracers to evaluate the contribution of fungal spores to organic carbon in atmosphere [16]. As the most abundant saccharide in soil of different areas, trehalose is used as a tracer for the resuspension of surface soil and unpaved road dust [17,18,19,20], while sucrose is the tracers for airborne pollen [16,21,22]. Much attention has been paid on characterizing these molecular tracers in atmosphere. Many techniques have been applied to determine carbohydrates in many fields [23]. The most frequently used one is liquid chromatography which is often combined with a variety of detectors, such as Evaporative Light Scattering Detector (ELSD) [23,24,25,26,27,28,29], Refraction Index (RI) [30,31,32], Ultraviolet Visible (UV) or fluorometric [33,34,35,36], high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [37,38], thin-layer chromatography (TLC) [39], charged aerosol detector [40]. Gas chromatography (GC) with flame ionization detection is also applied to quantify sugars in various fields [41,42,43,44]. However, due to the high limitation of detection they are not ideal for determining carbohydrates in aerosols. Therefore, researchers have made use of GC/MS to 3

determine these compounds in atmosphere, despite of shortcomings like that the pretreatment of samples is complex and time-consuming due to multistep derivatization procedures [2,16,23,45,46,47,48,49]. HPLC-MS/MS has been used to analyze carbohydrates in many fields during the past decade[48,50,51,52], whereas there are no highly acidic sites in chemical structures of carbohydrates. As a result, when performing atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), the ionization efficiency through deprotonation to form [M-H]- is relatively low [53,54]. Although [M-H]- has been used in LC-MS to determine carbohydrates in previous studies [52], the usage of conventional method to determine carbohydrates in atmosphere, which with a much lower concentration than biological or chemical samples, remains limited. How to improve the sensitivity of HPLC-MS/MS to analyze carbohydrates has been a challenge. Hence,

several

methods

have

been

adopted

such

as

that

derivatization

with

1,2-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one (PMP), which can be time-consuming when pretreating samples [23,55]. Apart from that, both post- and pre-column derivations are decent methods to improve sensitivities in HPLC-MS/MS. For example, in positive mode, [M+NH4]+ was monitored in SIM mode, with poor separation of these compounds [54,56]. Similarly, [M+Cl]- was also developed in SIM mode in negative mode [54,56,57,58]. Sodium adducts was also tried to use in our study, however, the results were not ideal. Because there was lack of sensitive and simple method to determine carbohydrates in the air, we validated a highly sensitive and selective HPLC-MS/MS method to determine carbohydrates in atmosphere by producing acetate adducts [M+CH3COO]-. The advantages of this method are that the limitation of detection is the lowest among all techniques quantifying carbohydrates and there is no need to derivative aerosol samples 4

to determine those compounds. 2 Experiment section 2.1 Chemicals Levoglucosan (98%), L-(-)-arabitol (99%), D-threitol (99%), (2R,3R) -2 -Methyl butane -1,2,3,4-tretraol (Canada, TRC), sucrose (reagent grade, 99%), D-sorbitol (99%), arabinose(99%), mannose (99%), galactose (99%), rhamnose (99%), xylose (99%) and ammonium salt (ammonium fluoride; mass spectrometry grade (≥99.99%, trace metals basis), ammonium acetate（≥99.0%, for mass spectrometry, eluent addictive for LC-MS）, ammonium chloride（99.998%, trace metals basis) ) were all purchased from Sigma-Aldrich. D-(+)- trehalose anhydrous (99%) and inositol (99%) were purchased from Macklin. Acetic acid glacial (high purity solvents) was obtained from TEDIA. Water (distilled water) was available to Watsons company. Methanol and acetonitrile (gradient grade for liquid chromatography) were obtained from Merck. 2.2 Aerosol sampling PM

2.5

filter samples were collected on the top of the Library building (32°03'N, 118°46'E,

~30 m above the ground), campus of Nanjing University of information Science & Technology (NUIST), Nanjing, China. The samples were collected on quartz microfiber filters (20.3 cm × 25.4 cm, Whatman) by a higher volume aerosol sampler (TISCH, USA). Samples were collected every 24 hours in 2017 with a flow of 1.13 m3/min. Seven blank samples were collected to examine possible contamination by placing quartz fiber filters on the holder when the sampler was turned off. All filters were pre-baked (about 4 h in a muffle furnace with 400°C) before sampling, then wrapped in tin foils after sampling and stored in -20 °C until analysis. 2.3 Pretreatment of aerosol samples 5

All glass wares were soaked in methanol for 24 h and dried before use. Ceramic scissor was cleaned with methanol. Aerosol samples were cut into 6 cm2 small pieces before placed into the glass ware. After ultrasonic extraction for 20 min with ice bath, the solvent was extruded through 0.22 μm filter and dried under N2 gently. The residual was redissolved in 500μL 6:4=acetonitrile : water and injected into analysis instrument. 2.4 Optimization of the LC-MS/MS procedure 2.4.1 LC conditions Carbohydrate analyses were performed with a Diane, U3000 high-performance liquid chromatograph. The 100 μL of sample solutions was injected into a Prevail Carbohydrate ES column (150 mm x 4.6 mm, 3 μm) with a flow rate of 300 μL /min, mobile phase is constituted with a 0.1% acetate acid water solution (solvent A) and a solution of acetonitrile (solvent D). The eluent program was isocratic elution A:D=4:6 running for 22 min. The 3:7= water: acetonitrile was also tested, however, this condition is not a good choice for quantifying large number of aerosol samples as it requires 45 minutes to run. Because several hours are required to spend on pretreatment of aerosol samples, we want to shorten the time of sample analysis and save organic solvent. 2.4.2 MS conditions The instrumental MS conditions were optimized by using standard compounds. We dissolved them respectively in methanol with 1μg ml-1 containing 0.05% acetate acid and infused into mass spectrometry at a flow rate of 10 μL/min directly. From what we have known, those adducts including 2-Methylbutane-1,2,3,4-tretraol have not been applied in previous studies. During the optimization, the parameters of ESI were set as follows: spray voltage: -2500 V, vapor temperature: 6

50 °C, sheath gas: 10 psi, aux gas: 0 psi, ion sweep gas: 0 V. In this section, center mass scan with width 40 was chosen to scan our parent ions and [M+CH3COO]- occurred in spectrometry. Tube lens value should be optimized at the first step. We found that all the parent ions had the highest abundance at a lower tube lens value compared with [M-H]- as shown in Table S1. At the second step, the collision energy was optimized to find the higher relative abundant daughter ions decomposed from [M+CH3COO]-. The results of parameters optimization were presented in Table S1 and Table 1. The most abundant transitions ([M+CH3COO]-→[M-H]-) were chosen to monitor in temporal selective reaction monitor scan mode to quantify these compounds. It was noted that the levoglucosan happened to have this transition of [M+CH 3COO]-→ 59. The fragmentation of [M-H]-, [M+H]+, [M+Na]+ were also explored in this study. 2.4.3 Selection of scan mode According to many previous studies [54,57,59], the select ion monitor (SIM) mode has always been applied to monitor parent compounds. In our study, SIM and temporal selected reaction monitor (t SRM) scan mode were both tried, and the sensitivities of different methods were exhibited in Table S2. However, some compounds coeluted in a similar time, which meant that one chromatographic peak could contain several compounds including both levoglucosan and inositol. Therefore, SIM scan mode was not the better choice. As observed, [M+CH3COO]- in SRM mode was the most sensitive methods. There were not all carbohydrates could form sodium adducts in ion source. But it was noticeable that [M+Na]+ occurred in three compounds including 2-methyltetrol, sucrose, trehalose which was barely reported before. 3 Results and discussion 3.1 The sensitivity of different scan methods 7

[M+H]+, [M+Na]+ in positive mode and [M+CH3COO]- , [M-H]- in negative mode have been all optimized and tested in this study. The most abundant parent ions observed in mass spectrometry were [M+CH3COO]- when direct injection. 1ppm of mixed standard was injected into separated under HPLC conditions to find the appropriate method to quantify carbohydrates. The responses shown in Table S2, indicated that the most sensitive and selective method was [M+CH3COO]- in t SRM method. 3.2 Matrix effect The assessment of reliability and matrix effect is crucial, when homologues are added as internal standards [60]. To evaluate the matrix effect of each compound through this method, three replicates of aerosol samples in different seasons were made to verify the absence of carbohydrates in atmosphere, then fortified with 500 μg L-1 mix standard solutions and internal standard to analyze. Matrix effects were evaluated by comparison of the peak areas of the fortified samples with ones from standard solutions of same concentration. The matrix effect occurred when the other compounds co-eluted with target compounds, because those compounds could enhance or suppress the ionization of target compounds in ESI source. In our study, the enhancement or suppression within 20% could be ignored. The main reason affecting matrix effect was that the components coeluting with target compounds. In general, purification and long analysis time of samples could decrease the matrix effect. The results of matrix effect were presented in Table 2. For levoglucosan, matrix effects in four seasons ranged from 104% to 118%, which could be herein regarded as no matrix effect. For 2-methlythretitol, the matrix effect ranged from 91% to 120% in four seasons, which could also been ignored. The matrix effects of sucrose were ignored as well, for the values ranged from about 8

80% to 110%. For threitol, arabitol and mannitol, these matrix effects ranged from 55% to 124% as illustrated in Table 2. Mannitol, there exited obviously matrix effects in the spring, summer, fall and winter with 67%, 69%, 81% and 55%. The matrix effects of inositol were also obvious in four seasons as well, which were 62%, 68%,65% and 72%, respectively as shown in Table 2. 3.3 Method validation The validation of this method was examined by evaluating linearity, selectivity, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day assay precision, recovery, accuracy and stability of these compounds [61]. 3.3.1 Selectivity The selectivity was evaluated through the quantification of various aerosol samples, which were compared with blank quartz filters spiked by the carbohydrate standard mix at 100 μg L-1. The method was considered to be specific because there was no significant interference at migration time, molecular ion and its fragments, as well as the ratio of relative abundance of fragment ions. All 10 determined carbohydrates could be distinguished from the other compounds in standard solution as demonstrated in Figure 1 (a), (b), (c) representing total ion chromatography and extract ion chromatography in standard solution. And Figure 2 (a), (b), (c) indicate those compounds in aerosol samples. The original chromatography maps were displayed in Figure S1 (a), (b), (c) and Figure S2 (a), (b), (c). 3.3.2 Linearity Internal method was used for quantification by comparing the target compound peak area with internal standards, namely 50 ng 13C levoglucosan. 1 μg L-1, 2 μg L-1, 5 μg L-1, 25 μg L-1, 50 μg L-1, 100 μg L-1, 250 μg L-1, 500 μg L-1, 1 μg L-1, 2 μg L-1, 5 μg L-1, 10 μg L-1, 25 μg L-1, 50 μg 9

L-1 compounds were prepared with addition of 500 μg L-1 internal standard to make standard curve. Linear equations, the determination of coefficients (R2) and linear ranges were obtained from the analytical curves for carbohydrates as summarized in Table 5. The R2 value was ranged from 0.991 to 0.999. 3.3.3 Limit of detection and limit of quantification The limit of detection (LOD) and limit of quantification (LOQ) were calculated as the lowest concentration to produce the ratio of signal to noise with 3 and 10. The LODs and LOQs of carbohydrates were presented in Table 3. The LODs of these carbohydrates ranged from 2 ng L-1 to 25 μg L-1, which was the most sensitive method comparing with other methods up to date shown in Table S3. 3.3.4 Precision Precision expressed by relative standard deviation (RSD) were calculated through quantifying five replicates of aerosol samples. The samples were added with different given amount carbohydrates mix (10, 100, 500 μg L-1) after extraction. Intra-day precision (repeatability) was calculated through the analysis of five triplicates and was carried out on the same day. While the inter-assay precision was determined by analyzing the same replicates in five different days. The precision was lower than 16% for all carbohydrates in both inter-day and intra-day case we studied. The results were presented in Table 4. 3.3.5 Recovery and stability Mixed standard carbohydrates with different concentration levels (10, 100, 500 μg L-1) were added into aerosol blank matrix samples to calculate recovery. Blank filters which substitute blank matrix because of lacking aerosol blank matrix were put on the highly volume sampler beforehand. 10

The recovery rate was determined by comparing the concentration of extracted samples (the amount added before extraction) with the amount added after extraction. The recoveries (Table 5) were between 85-115% with relative standard deviation (RSD) smaller than 15% for all carbohydrates. As for the carbohydrates stability, no significant decline (smaller than 1%) occurred in the target compounds signal from aerosol samples which were stored at -20 °C for 12 hours without contacting air and light. 3.4 Real sample analysis The validated method was used to the simultaneous quantification of 10 carbohydrates in 40 aerosol samples collected in suburban Nanjing in March, May, November and February. All carbohydrates in our study were found in all aerosol samples (Table 6) at the concentration from 0.12 ng m-3 to 607 ng m-3 in atmosphere. Figure 2 displayed the SRM chromatogram of aerosol samples with negative results for all carbohydrates. Due to the lowest LOQ values achieved by this developed method, it was possible to quantify carbohydrates with a trace amount of filter extracted without derivation, which could save time in analyzing a large amount of aerosol samples. 4. Conclusion Since the poor ionization efficiency of carbohydrates through deprotonation to form [M-H]in atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), the conventional HPLC-MS/MS method to determine those compounds in atmosphere is difficult. In this study, a highly sensitive method based on HPLC-MS/MS has been successfully developed to determine the content of carbohydrates in particulate matter. As far as we known, this is a highly 11

sensitive method to determine these compounds, while the preparation of aerosol samples is pretty simple. This may be the first time to choose the transition of [M+CH 3COO]-→[M-H]- as the quantification ions to determine carbohydrates. The analytical procedure has been validated through estimation of accuracy, precision and recovery, that can accurately quantify those compounds in atmosphere. Further study on highly sensitive HPLC-MS/MS method is still warranted in order to determine more compounds. Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Credit author statements

Wenjing Li, Mindong Chen, Xinlei Ge: Conceptualization, Methodology, data curation; Chuanxin Gu, Wentao Yu, Dongyang Nie: Data curation; Wenjing Li, Mindong Chen: Writing- Original draft preparation; Mindong Chen, Xinlei Ge: Writing- Reviewing and Editing; Mindong Chen: Supervision, Funding acquisition, Project administration.

Acknowledgements This work was supported by the National Key Research and Development Project（Grant No.2018YFC0213802）；and the National Natural Science Foundation of China (Grant No., 21577065, 21976094). Reference [1]

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Table 1 the parameters of optimized conditions of every compound with [M+CH3COO]-. Compounds

formula

tR (min)

Molecular

[M+CH3COO]-

Abundant

weight

Tube

Product

lens

ions

abundant

Collision energy

value Threitol

C4H8O3

2-methyltetrol

Arabitol

C5H12O4

C5H12O5

Levoglucosan 13

Levoglucosan C

12.95

11.61

14.07

122

136

152

181.1

195.1

211.1

7.06e5

5.49e5

7.02e5

41

44

42

59.2

1.84e5

16

121.0*

1.81e5

11

59.3

1.14e5

21

135.0

2.97e5

11

89.3

5.0e4

26

101.1

3.09e4

22

151.0*

4.52e5

13

C6H10O5

11.20

162

221.1

3.06e5

51

59.3*

2.66e5

15

13

11.20

168

227.1

4.52e5

48

59.3*

1.56e5

15

C6H10O5

34 Fructose

Glucose

Inositol

Sorbitol

Sucrose

Trehalose

C6H12O6

C6H12O6

C6H12O6

C6H14O6

C12H22O11

C12H22O11

14.05

16.25

20.48

20.48

16.25

17.77

180

180

180

182

342

342

239.1

239.1

239.1

241.1

401.2

400.9

17

1.45e5

1.54e5

9.42e4

3.19e5

2.35e5

2.87e5

33

49

54

32

51

52

59.2

3.94e4

23

89.1

5.87e4

16

178.9*

2.21e5

7

89.1

4.63e4

17

59.2

2.14e4

16

178.9*

1.40e5

7

160.9

1.61e4

21

195.0

6.06e4

19

178.9*

1.04e5

14

89.1

4.18e4

23

101.2

3.34e4

26

180.9*

6.16e5

13

118.9

4.73e4

28

178.8

6.77e4

22

340.6*

5.74e5

17

178.8

6.45e4

20

340.7*

4.53e5

15

89.1

3.46e4

28

Table 2. Matrix effects (%) of different carbohydrates in four seasons in this method. Matrix effects

levoglucosan

methythreitol

threitol

arabitol

sorbitol

Sucrose

trehalose

inositol

Spring

118

91

87

71

73

94

81

63

Summer

113

92

75

80

92

81

73

68

Fall

112

121

89

76

68

77

119

65

Winter

104

95

64

124

71

110

79

72

Table 3 Linearity, R2, LOD and LOD of different carbohydrates. Compounds

linearity

Conc. Range

R2

LOD

LOD

Threitol

Y=-0.004+0.002x

5-2000 μg L-1

0.993

1.0 μg L-1

3.0 μg L-1

2-methyltetrol

Y=-0.002+0.005x

1-2000 μg L-1

0.999

2.5 ng L-1

7.5 ng L-1

Arabitol

Y=0.023+0.036x

1-500 μg L-1

0.993

1.0 ng L-1

3.0 ng L-1

Levoglucosan

Y=0.005+0.002X

25-50000 μg L-1

0.999

25.0 μg L-1

75.0 μg L-1

Inositol

Y=0.003+0.014x

25-2000 μg L-1

0.991

5.0 ng L-1

15.0 ng L-1

Sorbitol

Y=0.094+0.033x

10-2000 μg L-1

0.992

0.5 ng L-1

1.5 ng L-1

Sucrose

Y=0.390+0.064x

25-2000 μg L-1

0.991

10.0 ng L-1

30.0 ng L-1

Trehalose

Y=0.020+0.080x

5-1000 μg L-1

0.997

2.0 μg L-1

6.0 μg L-1

Table 4 Intra-day precision and Inter-day precision. Compounds

Method Intra-day precision (RSD, %) 10 μg L

-1

100 μg L

-1

Inter-day precision (RSD, %)

500 μg L

-1

10 μg L-1

100 μg L-1

500 μg L-1

Threitol

13

7

7

4

2

1

2-methyltetrol

10

2

11

2

1

2

Arabitol

10

3

13

1

1

1

Levoglucosan

13

3

7

3

2

1

Inositol

10

5

9

3

1

1

Sorbitol

12

4

11

9

1

2

Sucrose

14

5

16

5

2

2

Trehalose

3

3

9

1

1

1

18

Table 5 Recovery of this method. Compounds

Spiked concentration levels 10 μg L-1 Rec

100 μg L-1

RSD (%)

(%)

500 μg L-1

2 mg L-1

5 mg L-1

Rec

RSD

Rec

RSD

Rec

RSD

Rec

RSD

(%)

(%)

(%)

(%)

(%)

(%)

(%)

(%)

111

4

109

3

88

8

101

1

Threitol

115

13

106

7

85

2

2-methyltetrol

107

12

102

4

99

2

Arabitol

84

14

101

5

92

4

Levoglucosan

113

4

115

15

113

1

Inositol

102

14

90

4

95

3

Sorbitol

120

14

110

6

102

1

Sucrose

102

2

114

8

99

5

Trehalose

116

13

98

10

78

2

Table 6. The concentration of carbohydrates in atmosphere (unit: ng m-3, NA: not detect). Date

levoglucosan

2-methythreitol

threitol

arabitol

sorbitol

Sucrose

trehalose

Inositol

MARCH10

581.09

NA

NA

2.65

NA

10.25

0.30

NA

MARCH11

141.27

NA

0.26

0.91

1.64

3.98

1.43

1.08

MARCH12

149.42

NA

0.60

0.85

0.80

1.51

0.48

0.79

MARCH14

431.66

0.29

0.87

2.36

2.38

4.62

1.73

2.75

MARCH17

221.41

0.46

0.51

1.08

1.18

3.68

0.88

1.34

MARCH18

385.29

0.60

0.84

1.92

2.53

5.08

1.24

2.39

MARCH2

779.87

0.51

NA

2.97

1.30

0.38

1.39

3.15

MARCH21

597.95

0.82

2.89

16.37

16.47

11.58

3.48

8.37

MARCH25

228.88

0.26

0.51

1.49

2.19

3.63

1.46

2.12

MARCH26

414.37

0.35

0.75

5.57

5.37

2.57

0.97

2.91

MAY10

369.12

3.81

1.32

9.29

15.19

11.28

9.97

3.78

MAY11

110.28

2.86

NA

2.49

3.78

4.26

1.74

1.33

MAY12

66.00

2.50

0.23

1.91

4.47

5.64

2.36

0.99

MAY13

85.46

1.55

0.83

10.82

11.96

11.98

4.83

3.25

MAY14

289.83

1.94

1.00

10.40

13.15

52.36

6.29

5.82

MAY15

114.77

1.02

NA

2.25

3.13

11.56

1.79

1.57

MAY16

180.20

1.62

NA

2.77

3.44

9.86

NA

3.20

MAY17

126.43

1.99

NA

2.88

4.12

8.79

5.29

2.26

19

MAY18

133.50

4.26

0.28

3.53

5.39

12.10

12.24

2.01

MAY19

115.59

2.41

0.19

1.61

1.66

2.40

3.99

NA

NOV20

316.42

0.51

0.78

1.96

1.62

0.76

0.98

1.98

NOV21

867.26

0.55

NA

7.17

6.98

1.31

2.80

3.17

NOV22

446.72

0.48

1.05

2.20

1.95

1.39

1.25

2.40

NOV23

946.81

NA

3.13

12.27

11.65

3.77

5.27

5.52

NOV24

1105.98

1.15

3.94

16.56

17.19

3.54

4.76

9.85

NOV25

1864.44

0.91

3.06

12.66

9.75

1.65

2.46

6.06

NOV26

574.06

0.58

1.62

5.07

4.69

0.98

1.66

2.57

NOV27

146.48

1.08

0.59

3.63

5.53

17.43

2.98

2.27

NOV28

948.87

1.15

1.90

4.66

2.90

1.64

1.77

4.88

NOV29

329.26

0.38

0.86

2.49

2.48

0.85

1.02

1.53

JAN132018

903.39

0.73

1.22

3.56

3.85

4.70

3.18

4.73

JAN142018

1027.75

1.06

1.18

4.03

3.96

5.87

3.29

6.77

JAN152018

382.63

0.37

NA

1.14

1.06

1.69

3.17

1.91

JAN162018

719.37

0.72

NA

2.49

1.49

1.48

1.33

3.83

JAN172018

500.32

0.80

0.73

1.36

0.94

0.80

1.20

2.81

JAN182018

313.75

0.52

0.35

1.04

0.71

0.75

1.06

1.63

JAN192018

610.01

1.05

0.98

1.68

1.31

0.85

1.97

2.70

JAN202018

588.99

0.74

NA

1.85

1.32

7.21

1.10

3.01

JAN212018

266.71

0.33

NA

0.81

0.70

0.92

0.98

1.23

2000 1500

levoglucosan

1000 500

40

2-Methylbutane-1,2,3,4-tretraol

20

0 6000

arabitol

4000

2000

x10

3

x10

3

intensitiy

x10

3

0 60

0 20 15 10 5 0 120

threitol

80

TIC

40 0

0

5

10 Time(min)

(a)

20

15

20

trehalose

3

120

sucrose

x10

80 40

0 3 30x10

sorbitol

intensity

20 10

x10

3

0 12

inositol

8 4

13

levoglucosan

C

x10

3

0 20 15 10 5 0

5

10 Time(min)

15

20

(b)

40 30 20 10 0 50 40 30 20 10 0 2500 2000 1500 1000 500 0 25 20 15 10 5 0 600

x10

3

xylose

10

5

10

5

10

5

10

15

20

25

30

20

25

30

20

25

30

20

25

30

galactose 15

3

arabinose 15

x10

intensitive

x10

3

rhamnose 5

mannose15

400 200

0

5

10 Time(min)

15

20

(c) Figure 1. The chromatographic separation of carbohydrates in standard solution.

21

50 40 30 20 10 0 2500 2000 1500 1000 500 0 16

intensity

x10

3

2-Methylbutane-1,2,3,4-tretraol

arabitol

x10

3

12

threitol

8

3

4 0 120

TIC

x10

80

40 0

0

5

10

Time(min)

15

20

25

x10

3

x10

3

(a) 40 30 20 10 0 20 15 10 5 0

sucrose sorbitol

3

inositol

30x10 20

intensity

trehalose

10 0

x10

3

4000 3000 2000 1000 0 120

13

levoglucosan

C

levoglucosan

80 40 0

0

5

10 Time(min)

(b)

22

15

20

25

x10

3

15

galactose

10 5

0 6000 4000 2000

mannose

intensitive

0

5

1200

10

xylose

15

20

25

30

15

20

25

30

20

25

30

800 400 0

x10

3

120

5

80

10

rhmnose

40 0 800

5

600

10

arabinose 15

400 200

0

5

10

15

20

Time(min)

(c) Figure 2. The chromatographic separation of carbohydrates in aerosol samples on March 1.

23