Determination of sulfonamides in blood using acetonitrile–salt aqueous two-phase extraction coupled with high-performance liquid chromatography and liquid chromatography–tandem

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AnalyticalMethodsAdvancing Methods and Applications

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umber 1 | 2010

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1

Determination of sulfonamides in blood using acetonitrile-salt 1

aqueous two-phase extraction coupled with high-performance liquid 2

chromatography and liquid chromatography–tandem mass 3

spectrometry 4

Wei Yub Zhongling Liua Shiqian Gaoa Shusen Cuib Xiao Yanga 5

Wei Qiuc Hanqi Zhanga Aimin Yua Yanfu Huana,* 6

a College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China 7

b Department of Hand Surgery, China-Japan Union Hospital, Jilin University, Xiantai Street 126, Changchun 8

130033, PR China 9

c Hepatobiliary and Pancreatic Surgery, The Bethune First Hospital , Jilin University, Xinmin Street No. 71, 10

Changchun 130033, PR China 11

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*Corresponding author. Yanfu Huan 20

Tel.: +86 431 85168399; fax: +86 431 85112355. 21

E-mail address: [email protected] 22

Page 1 of 27 Analytical Methods

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View Article OnlineDOI: 10.1039/C3AY40902C


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Abstract 23

Acetonitrile-salt aqueous two-phase extraction system (ATPS) was developed and 24

applied for the extraction of sulfonamides in blood. HPLC and HPLC-MS were 25

applied to the determination of the analytes. 3 mL of blood sample was diluted with 1 26

mL purity water and 6 mL of acetonitrile and 1 g of K2HPO4 were added into the 27

sample. The mixture was ultrasonically shaken for 5 min. The ATPS was formed. The 28

hemoglobin in blood was gathered together and suspended between acetonitrile and 29

aqueous phases. The parameters affecting the extraction efficiency, such as type and 30

volume of extraction solvent, type and amount of salt, pH value of sample solution, 31

extraction time and the amount of dissolution solvent were investigated. The limits of 32

detection for sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole and 33

sulfafurazole were 4.81, 1.87, 2.73, 2.17 µg L-1, respectively. When the present 34

method was applied to the analysis of blood samples, the recoveries of the analytes 35

ranged from 80.1 to 96.9 % and relative standard deviations were lower than 7.13 %. 36

37

Key words: Acetonitrile-salts aqueous two-phase extraction; sulfonamides; blood; 38

drug metabolism; HPLC-MS 39

40

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Page 2 of 27Analytical Methods

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1. Introduction 45

46

The sulfonamides (SAs) are synthetic bacteriostatic drugs which are effective 47

against many gram-positive and many gram-negative bacteria. The SAs are used in 48

animal husbandry, aquaculture and also used as medicines to treat many kinds of 49

infection and then released to the environment after a series of metabolic [1,2]. The 50

overuse of SAs can build up the resistance of pathogenic bacteria to the drugs. The 51

SAs often work in conjunction with other compounds to stimulate their activities [3]. 52

The resistance of the bacteria to a kind of SA drug can result in the resistance to all of 53

the SA drugs [4]. However，more and more SAs are overused, which makes bacteria 54

build up resistance to the SAs. 55

In the past decades, various methods have been employed to determine SAs, such 56

as liquid chromatography–mass spectrometry (LC–MS) [5,6], high-performance 57

liquid chromatography ultraviolet detection (HPLC-UV) [7,8] and fluorescence 58

detection [9]. GC-MS is not suitable for SAs due to the low volatility of SAs. LC-MS 59

has proven to be a promising technique because the method has high selectivity, 60

specificity and sensitivity for the determination of SAs [10,11]. Up to now, LC-MS 61

was rarely reported to detect SAs in blood [12,13]. 62

Aqueous two-phase system (ATPS) is a powerful extraction technique and rapidly 63

developed in recent years [14]. ATPS, such as water–organic solvent [15,16,17] or 64

ionic liquid [18] was applied. 65

In this work, acetonitrile was used as the extraction solvent. The ATPS was formed 66


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in the presence of the inorganic salt. The ATPS can be applied for the extraction and 67

enrichment of SAs in blood. 68

69

2. Experimental 70

71

2.1. Reagents and chemicals 72

73

Sulfamonomethoxine (SMZ), sulfachlorpyridazine (SCPD), sulfamethoxazole 74

(SMO) and sulfafurazole (SFZ) were obtained from Sigma-Aldrich Chemical 75

Company (St. Louis, MO, USA). The purity of all the compounds was higher than 76

98 %. The structures and properties of these SAs are shown in Fig. 1 and Table 1, 77

respectively. Standard stock solutions for the SAs at the concentration level of 500 µg 78

mL−1 were prepared in acetonitrile containing 5.24 mmol L−1 acetic acid. The working 79

solutions were obtained by diluting the stock solutions with pure water. Formic acid 80

was of analytical-reagent grade and purchased from Beijing Chemical Factory 81

(Beijing, China). Anhydrous di-potassium hydrogen phosphate was purchased from 82

Aladdin Chenistry Co.Ltd (Nanqiao Town, Fengxian District, No.1008 QIGANG 83

Road in Shanghai). Chromatographic grade acetonitrile was purchased from Fisher 84

Scientific Company (UK) and pure water was obtained with a Milli-Q water 85

purification system (Millipore Co., USA). 86

87

2.2 . Instruments 88


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89

The 1100 series liquid chromatograph (Agilent Technologies Inc., USA) equipped 90

with UV detector and quaternary gradient pump was used. Zorbax Eclipse Plus-C18 91

column (150mm×4.6 mm, 3.5 µm, 162 Agilent, USA) and a C18 guard column (7.5 92

mm× 2.1 mm I.D., 5 µm) were used. In the optimial experimental conditions 93

HPLC-UV system was used. 94

All HPLC–MS experiments were carried out on an Agilent Technologies 1100 95

HPLC system (Palo Alto, CA, USA) coupled with an ABIQ-Trap mass spectrometer 96

(Applied Biosystems Sciex, Foster City, USA) equipped with ESI source. The 97

instrument was connected with a computer running Applied Biosystems Analyst 98

Version 1.4 software. Mass spectrum data were obtained with Bio-Analyst software 99

(Applied Biosystems, Sydney, Australia). 100

In the evaluation of analytical performance and application of the present method 101

the HPLC-MS system was used. The KQ-100DE ultrasonic cleaner was purchased 102

from Kunshan Ultrasonic Instrument Co., Ltd. (Kunshan, China). The frequency and 103

output power of the ultrasonic cleaner are 40 kHz and 100 W, respectively. RE-52AA 104

vacuum rotator evaporator (Yarong, Shanghai, China) was used. 105

106

2.3. Samples 107

108

Fresh human blood sample (Sample 1) was obtained from local hospital. Rabbit 109

blood sample (Sample 2) was obtained from local animal experiment center. Pig 110


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(Sample 3), cow (Sample 4) and chicken (Sample 5) blood samples were respectively 111

obtained from local market. The samples were stored at -20 ◦C before use. Except for 112

the experiments mentioned in Section 3.3.1, which were performed with all five 113

samples, all other results were obtained with Sample 1. The spiked samples 114

containing SAs were prepared by spiking the working solutions into blood samples 115

and stored at 4 ◦C for one week. The samples used for pharmacokinetic investigation 116

were described in Section 3.4. 117

Five male Japan big-eared white rabbits 2.5-3.2 Kg, 5 months old were provided by 118

laboratory animal centre of Jilin University（License NO. SCXK（Ji）2009-0003）. All 119

animals were kept from birth under housing conditions (temperature: 22 ± 1°C; 120

relative humidity, 60 ± 3 %; 12-hour light/darkness cycle; low noise disturbances), 121

allowing free access to a standard diet in pellets with water. All experimental 122

protocols were in accordance with the guidance suggestions for the Care and Use of 123

Laboratory Animals, formulated by the Ministry of Science and Technology of China124

（The Ministry of Science and Technology of the Peoples Republic of China. 125

Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30）. 126

Heparin saline（ 12500u Heparin： 1000ml 0.9 % saline； Hebei Changshan 127

Biochemical Pharmaceutical Co. Ltd., Shijiazhuang, China), gavage needle, and 128

sulfamethoxazole Tables(Shanghai New Asia Pharmaceutical Co. Ltd., Shanghai, 129

China) were used. 130

131

2.4. HPLC-UV conditions 132


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133

The mobile phase consists of acetonitrile (2.67 mmol L-1 formic acid) (A) and 134

aqueous solution (2.67 mmol L-1 formic acid) (B). The gradient program is as follows: 135

0–10 min, 10–30 % A; 10–20 min, 30 –34 % A; 20–21 min, 34–35 %A; 21–24 min, 136

35 % A and 24–30 min, 35–10 % A. The flow rate of mobile phase was kept at 0.5 mL 137

min−1 and column temperature was kept at 40 ◦C. The injection volume of analytical 138

solution was 20 µL. The monitoring wavelength was 270 nm. 139

140

2.5. HPLC- MS conditions 141

142

The HPLC conditions are the same as these mentioned above. MS conditions were 143

optimized. The ionspray voltage was held at 3.5 kV. The source temperature and 144

desolvation temperature were 120 ◦C and 380 ℃ respectively. Nitrogen was used as 145

cone gas. The flow rates of the cone gas 1 and 2 were 50 L hr-1 and 60 L hr-1, 146

respectively. 147

The precursor-product ion pair, declustering potential (DP), entrance potential (EP), 148

collision cell entrance potential (CEP), collision energy (CE) and collision cell exit 149

potential (CXP) were optimized with direct injection of the standard solution and are 150

listed in Table 2. The multiple reaction monitoring (MRM) mode was applied. 151

152

2.6. Extraction procedure 153

154


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3 mL of blood sample was placed into 10 mL Centrifuge tube and diluted with 1mL 155

of purity water. 6 mL of acetonitrile and 1 g of K2HPO4 were added into the tube. 156

Because KH2PO4 was dissolved slowly and existed in solid form for a long time, 157

K2HPO4 was used. The pH value of the resulting mixture was adjusted to 5.0 with 0.1 158

mol L-1 HCl. 159

The mixture was ultrasonically shaken for 5 min. The ATPS was formed. The 160

resulting solution was centrifuged at 5 ℃ for 10 min at 15,000 rpm and the 161

hemoglobin was suspended between the acetonitrile and aqueous phase. 5 mL of the 162

upper acetonitrile phase was placed in 10 mL centrifuge tube. Then acetonitrile was 163

evaporated to dryness under reduced pressure at 40 ◦C. The residue was dissolved in 164

500 µL of acetonitrile containing 2.67 mmol L-1 formic acid. The resulting solution 165

was filtered by 0.22 µm PTFE filter membrane before analysis. 166

167

3. Results and discussion 168

169

3.1. Optimization of extraction conditions 170

171

3.1.1 Selection of salts 172

173

The addition of salt is beneficial to the phase separation for some micellar systems, 174

since the density of the bulk aqueous phase increases [19,20]. As shown in Fig. 2, 175

ATPS can be formed in the presence of K2HPO4 and the hemoglobin is suspended 176


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between the acetonitrile and aqueous phase. However, the ATPS cannot formed in the 177

absence of K2HPO4. The effect of inorganic salts K2HPO4, NaCl, Na2CO3 and 178

(NH4)2SO4 were investigated. NaCl can not drive the system to separate into two 179

phases. Blood sample turns green after adding Na2CO3, the recoveries and peak shape 180

are affected. The peak shape in chromatogram in the presence of K2HPO4 and Na2CO3 181

are shown in Fig. 3. The recoveries obtained with (NH4)2SO4 are lower than those 182

obtained with K2HPO4. So K2HPO4 was selected. 183

184

3.1.2 Amount of K2HPO4 185

186

The effect of K2HPO4 amount was investigated. When 2.5 g of K2HPO4 was used, 187

the peak shape was unsatisfactory and when 1.0 g of K2HPO4 was used the highest 188

recoveries of analytes were obtained. So the affect of amount of K2HPO4 (0.8-1.3 g) 189

was investigated. The detailed information can be found in Fig. 4. Based the 190

experimental results, the amount of K2HPO4 was selected as 1.0 g. 191

192

3.1.3 Selection of organic solvents 193

194

Four organic solvents, including acetonitrile, methanol, ethanol and acetone, were 195

used as extraction solvents. When methanol and ethanol were used ATPS was not 196

formed in the presence of K2HPO4. The extraction recoveries obtained with 197

acetonitrile dramatically higher than those obtained with acetone. When acetone was 198


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used as extraction solvent, the recoveries of SMM, SCPD, SMO, SFZ are 14.86 %, 199

17.74 %, 13.93 %, 16.94 %, respectively. So acetonitrile was used as the extraction 200

solvent in the following experiments. 201

202

3.1.4 Volume of acetonitrile 203

204

To evaluate the effect of the volume of acetontrile on the determination of SAs, a 205

series of experiments were performed by adding different volume of acetonitrile (4–8 206

mL). The recoveries of the analytes increase with the increase of acetonitrile volume 207

from 4 to 6 mL. When the volume of acetonitrile increases from 7 to 8 mL, the 208

recoveries of analytes decreases. The detailed information can be found in Fig. 5. This 209

reason may be that hemoglobin can be rapidly denatured and gathered together in the 210

presence of a large amount of acetonitrile and SAs can be embedded in the 211

hemoglobin. Therefore, 6 mL was selected as the optimal volume of acetonitrile. 212

213

3.1.5 Effect of pH 214

215

SAs are insoluble in water and soluble in acidic media, So the pH value has a 216

significant effect on the extraction recoveries of the analytes. The effect of pH value 217

of the sample solution in the range of 2.0-10.0 on the extraction recoveries was 218

studied. The detailed information can be found in Fig. 6. The highest extraction 219

recoveries are obtained at pH 5.0. When the pH value of solution was higher than 7.0, 220


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the hemoglobin can not gathered completely and the extraction recoveries are low. At 221

low pH, SAs would be protonated, which renders them soluble in the aqueous phase. 222

Therefore, the pH value of 5.0 was optimal. 223

224

3.1.6 Effect of extraction time 225

226

Theoretically, the increase of extraction time (sonication time) was beneficial to the 227

partition equilibrium of target analytes in acetontrile/H2O system and the 228

improvement of the recoveries of analytes. The effect of extraction time was 229

investigated. The recoveries of analytes increase sharply with the increase of the 230

extraction time. When the time is longer than 5 min, the recoveries of analytes 231

decrease slightly. The experimental results indicate that the extraction equilibrium 232

could be achieved within 5 min. The detailed information can be found in Fig. 7. The 233

extraction equilibrium can be achieved in short time and the phase-transfer of the 234

target analytes is fast. Therefore, the extraction time of 5 min was selected. 235

236

237

3.2. Analytical performances 238

239

SAs were determined under the above optimized conditions. Fig. 8 shows LC–MS 240

chromatograms of the blood sample. 241

The working curves were constructed by plotting the peak areas measured versus 242


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the concentrations of analytes in the spiked samples. The slope and intercept of the 243

linear regression equations, the residual standard deviations (Sy/x) and correlation 244

coefficients for HPLC-MS are listed in Table 3. The experiment limits of detection 245

(ELODs) and quantification (ELOQs) for HPLC-MS indicated in Table 3 are 246

determined as the lowest concentrations yielding a signal-to-noise (S/N) ratio of 3 and 247

10, respectively. The concentrations of the target analytes in the extract are higher 248

than the ELODs and lower than upper limits of determination for the proposed 249

method. So the ELOQs and linear regression equations are appropriate to the goal of 250

the present method. 251

The slope and intercept of the linear regression equations, the residual standard 252

deviations (Sy/x) and correlation coefficients for HPLC are listed in Table 4. The 253

experiment limits of detection (ELODs) and quantification (ELOQs) for HPLC 254

indicated in Table 4 are determined as the lowest concentrations yielding a 255

signal-to-noise (S/N) ratio of 3 and 10, respectively. The working solutions of SAs at 256

the concentration of 20, 40, 80, 160 and 320 µg L-1 were directly injected to the 257

system and analyzed. The standard curves were obtained. Based on the standard 258

curves. The HPLC instrument limits of detection (ILOD) for SMZ, SCDP, SMO, SFZ 259

indicated are determined as the lowest concentrations yielding a signal-to-noise (S/N) 260

ratio of 3 and shown in the Table 4. The enhancement factors were determined by 261

comparing the slope of the calibration curve with and without pre-concentration, 262

respectively. The enhancement factor of the SMM, SCDP, SMO and SFZ were 5.32, 263

5.10, 5.44, 5.40, respectively. 264


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Repeatability was evaluated by determining target analytes in spiked blood samples. 265

The intra-day precision was determined by analyzing the samples five times in one 266

day. The inter-day precision was achieved by analyzing the samples once a day in five 267

consecutive days. The results for HPLC-MS are presented in Table 3 and indicate that 268

the present method has good repeatability. Long-term stability of analytes in blood 269

during sample storage was evaluated. The spiked samples were prepared according to 270

the method mentioned in Section 2.3, kept in sealed heparin tube and then stored for 1, 271

10, 20 and 30 days at 4 ◦C, respectively. All experiments were performed in five 272

replicates. The results are listed in Table 5. The recoveries and RSD values range from 273

80.6 to 96.1 % and 0.5 to 6.9 %, respectively. It can be concluded that the SAs in the 274

blood samples were stable for at least one month. 275

276

3.3. Application of the present method 277

278

3.3.1 Aanlysis of samples 279

In order to evaluate the applicability of the present method, this method was 280

applied to the determination of the residues of the SAs in blood. The results are listed 281

in Table 6. It can be seen that the present method provides good recoveries 282

(80.1-96.9 %) and acceptable precision (≤ 7.1 %). 283

284

3.3.2 Pharmacokinetic investigation 285

286


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Five male Japan big-eared white rabbits 2.5-3.2 Kg, 5 months old were provided by 287

laboratory animal centre of Jilin University（License NO. SCXK（Ji）2009-0003）. All 288

animals were kept from birth under housing conditions (temperature: 22 ± 1°C; 289

relative humidity, 60 ± 3 %; 12-hour light/darkness cycle; low noise disturbances), 290

allowing free access to a standard diet in pellets with water. All experimental 291

protocols were in accordance with the guidance suggestions for the Care and Use of 292

Laboratory Animals, formulated by the Ministry of Science and Technology of China293

（The Ministry of Science and Technology of the Peoples Republic of China. 294

Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30）. 295

Heparin saline（12500 u Heparin：1000ml 0.9 % saline； Hebei Changshan 296

Biochemical Pharmaceutical Co. Ltd., Shijiazhuang, China), gavage needle, and 297

sulfamethoxazole Tables(Shanghai New Asia Pharmaceutical Co. Ltd., Shanghai, 298

China) were used. 299

Before the experiment, five male rabbits were starved for at least 12h, but water 300

was given ad libitum. Oral doses (nominal 130 mg kg-1 body weight) of SMO was 301

administered by syringe as a solution in distilled water (25–35 mL). After dosing, 302

rabbits were housed singly in stainless-steel cages，and 3mL blood samples were 303

collected from ear vein in 2 mL centrifuge tube after oral administration for 0, 0.25, 304

0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48 h, respectively. All the blood samples were 305

stored at -20℃ until analyzed. The curve of SMO concentration in plasma - time is 306

shown in Fig. 9. The Cmax of SMO is 84.3 µg mL-1 appearing about at 1 h. 307

308


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4．．．．Conclusion 309

310

The ATPS was successfully applied to the extraction of the SAs from blood 311

samples. In the acetonitrile-aqueous-K2HPO4 solution the hemoglobin can be gathered 312

together and suspended between phases. The hemoglobin was not removed from the 313

sample by pretreatment methods, such as centrifugation. The analytes were extracted 314

into the organic phase. The method is simple and convenient. So it seems possible to 315

extend this method to the extraction of SAs in other similar samples by varying the 316

extraction conditions. 317

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References 331

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[2] P.Huovinen, L.Sundstrom, Antimicrob Ag Chemother, 1995 (39) 279–289. 334

[3] G. Castan˜eda Penãlvo, F.J. Guzmań Bernardo, J. Chromatogr. A, 2000 (870) 169–177. 335

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[5] Anna Bialk-Bielinska, Jolanta Kumirska, Talanta, 2009 (80) 947-953. 337

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359

360

361

362

363

364

365

366

367

368

369

370

371

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18

Figure captions 375

376

Fig. 1. Chemical structures of (a) SMM; (b) SCPD; (c) SMO; (d) SFZ. 377

Fig. 2. Phase separation in the absence (a) and presence (b) of K2HPO4 378

Fig. 3. Peak shape in HPLC chromatograms of spiked sample in the presence of 379

K2HPO4 (a) and Na2CO3 (b). 1, SMM ; 2, SCPD; 3, SMO; 4, SFZ; 380

Fig. 4. Effect of K2HPO4 amount. 381

Volume of acetonitrile, 6 mL; sample amount, 1 mL; H2O, 3 mL; extraction time, 5 382

min; dissolution volume,500 µL. 383

Fig. 5. Effect of acetonitrile volume. 384

Sample amount, 1 mL;H2O, 3 mL ; K2HPO4 amount, 1 g; extraction time, 5 min; 385

dissolution volume,500 µL. 386

Fig. 6. Effect of pH. 387

Volume of acetonitrile, 6 mL; sample amount, 1 mL; H2O, 3mL; K2HPO4 amount, 1 g; 388

extraction time, 5 min; dissolution volume,500 µL. 389

Fig. 7. Effect of extraction time. 390

Volume of acetonitrile, 6 mL; sample amount, 1 mL;H2O, 3 mL; K2HPO4 amount, 1 g; 391

dissolution volume,500 µL. 392

Fig. 8. LC–MS/MS chromatograms of spiked sample.1, SMM ; 2, SCPD; 3, SMO; 4, 393

SFZ; 394

Fig. 9. Curve of SMO concentration in plasma - time 395

Volume of acetonitrile, 6 mL; sample amount, 3 mL;H2O, 3 mL; K2HPO4 amount, 1 g; 396


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19

extraction time, 5 min; dissolution volume, 500 µL. 397

398


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Fig. 1


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Fig. 2

a

b

Fig. 3


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2 4 6 8 10

0

20

40

60

80

100

Recovery (%)

PH

SMM

SCPD

SMO

SFZ

Fig. 4

Fig. 5

4 5 6 7 8

0

20

40

60

80

100

Recovery (%)

Acetonitrile Volume (mL)

SMM

SCDP SMO SFZ


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2 4 6 8 10

0

20

40

60

80

100

Recovery (%)

PH

SMM

SCPD

SMO

SFZ

Fig. 6

0 2 4 6 8 10 12 14 16

50

60

70

80

90

100

Recovery

(%

)

Extraction time (min)

SMM

SCPD

SMO

SFZ

Fig. 7


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Fig. 8

Fig. 9


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Table1

Physicochemical properties of SAs

SAs Molecular weight PKa1 PKa2 LogKw0

SMM 281.2 1.42 6.67 0.70

SCDP 285.1 1.88±0.50 5.90±0.30 0.32

SMO 254.2 1.52 4.83 1.01

SFZ 268.1 1.85±0.30 5.60±0.04 0.89

Table2

Ion pair and selected operation parameter for MS

SAs Ion pair Declustering

potential (DP)

Entrance

potential (EP)

Collision cellentrance

potential (CEP)

Collision

energy (CE)

Collision cellexit

potential (CXP)

SMM 281.2/215.2 30 6.0 40 25 4.0

SCPD 285.1/156.1 30 6.0 30 20 2.0

SMO 254.2/160.2 35 8.0 30 20 4.0

SFZ 268.1/156.1 35 6.0 50 20 2.0

Table3

Regression equations, LODs and LOQs for HPLC-MS

Compo

und

Regression

equation

(n=5)

Corrlation

coefficient

Linear

range(μg·L-1

) Sy/x

ELOD

(μg·L-1

)

ELOQ

(μg·L-1

)

Intra day

precision

(RSD, %, n=5)

Inter day

precision

(RSD, %, n=5)

SMM 0.9998 A=28811C-278 12.9-320.1 28419 4.81 16.03 2.4 2.2

SCPD 0.9999 A=33722C+2595 8.4-274.0 11683 1.87 6.23 1.7 3.8

SMO 0.9996 A=38452C+2038 9.6-230.4 31005 2.73 9.10 2.7 5.0

SFZ 0.9999 A=34542C+1172 10.1-266.9 21852 2.17 7.23 2.0 4.9

C: µg. mL-1

Table4

Regression equations, LODs and LOQs for HPLC for HPLC

Compoun

d

Regression

equation

(n=5)

Corrlation

coefficient Linear range(μg·L

-1) Sy/x

ELOD

(μg·L-1

)

ELOQ

(μg·L-1

)

ILOD

(μg·L-1

)

SMM 0.9996 A=0.7185c-6.08 8.3-266.7 29.5 4.5 14.9 11.11

SCPD 0.9993 A=0.7828c+21.82 7.7-245.1 35.9 3.8 12.8 9.85

SMO 0.9999 A=0.8927c-4.51 6.7-214.8 13.58 3.3 7.53 9.15

SFZ 0.9995 A=0.8091c+8.26 7.4-238.3 35.36 3.6 12.1 10.03

C: µg. L-1


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Table5

The recoveries of the analytes in spiked sample 1

Add

(μg.L-1)

Stored

time

(weeks)

SMM SCDP SMO SFZ

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

50 1 85.2 2.4 83.6 3.3 90.0 5.2 86.5 6.0

2 87.7 1.8 89.9 1.4 86.7 2.4 90.6 5.0

3 92.2 6.4 91.7 2.9 92.4 3.1 91.3 1.7

4 88.9 1.9 81.6 6.9 95.8 3.7 87.1 5.6

6 90.3 4.4 88.0 4.3 87.9 2.0 93.2 2.7

8 84.9 4.9 89.2 2.1 93.4 4.2 84.9 2.5

150 1 91.2 2.3 83.5 6.4 91.5 0.9 89.0 6.1

2 88.8 2.5 80.6 4.2 96.1 5.2 85.7 3.5

3 92.4 1.8 84.8 1.9 87.0 3.6 92.9 1.7

4 85.1 5.7 90.2 3.3 90.2 2.6 93.1 3.6

6 89.2 6.2 82.9 1.9 91.5 2.5 88.2 0.5

8 82.7 3.6 87.4 5.3 86.2 1.3 84.3 2.5

Table6

Analytical results of blood samples (n=5)

Sample Added (μg.

L-1

)

SMM SCDP SMO SFZ

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

Recover

y(%)

RSD(%

n=5)

Sample 1 50 91.3 6.9 83.6 3.3 88.2 6.0 87.6 2.3

150 85.7 6.6 89.7 4.7 94.5 1.6 80.9 3.3

Sample 2 50 85.2 3.5 87.3 4.0 91.4 3.9 89.1 4.9

150 89.6 4.4 90.6 3.8 96.9 6.2 80.7 2.6

Sample 3 50 90.3 3.8 80.1 3.6 84.8 2.7 85.1 5.2

150 92.4 2.3 87.2 5.6 89.1 6.7 90.5 1.8

Sample 4 50 88.5 1.0 82.5 4.8 95.3 3.9 82.1 1.6

150 81.1 3.2 90.3 2.2 93.6 4.1 88.5 4.8

Sample 5 50 95.2 5.3 82.3 1.5 82.6 7.1 86.4 2.9

150 86.7 5.5 89.4 2.5 96.3 5.3 90.2 5.4


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