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Analytical and Bioanalytical Chemistry

Electronic Supplementary Material

Silica based 2-(N,N-dimethylamino)-1,3-propanediol hydrophilic

interaction liquid chromatography stationary phase for separating

cephalosporins and carbapenems

Wei Yin, Lingping Cheng, Huihui Chai, Ruiqiang Guo, Renhua Liu, Changhu Chu,

John A. Palasota, Xiaohui Cai

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Experimental section

Chemicals and Reagents

Spherical silica (5μm partical size; 10 nm pore size; 300 m2 g-1 surface area) was

purchased from Fuji Silysia Chemical Ltd. (Japan). HPLC grade actetonitrile (ACN),

formic acid and methanol were from Sigma (USA) and Tedia (USA) respectively.

Milli-Q ultrapure water was used throughout the HPLC experiment.

1-(p-nitrophenyl)-2-N,N-dimethylamino-1,3-propanediol, imidazole, sodium azide,

acetylchloride, CuSO4.5H2O, ammonium formate and sodium ascorbate were all

domestic reagent(National Medicine Corporation Chemical Reagent Ltd., Shanghai,

China) and used without further purification. 3-isocyanatopropyl-triethoxysilane and

propargylamine (National Medicine Corporation Chemical Reagent Ltd., Shanghai,

China) were purified by distillation before use. Solvents of N-dimethyl formamide

(DMF), methanol and ethanol (Ninfeng Chemical Reagent Ltd., Shanghai, China)

were used as received. The test solutes used for HILIC evaluation were commercially

available test compounds such as uracil, 5-methyluridine, adenine, cytosine,

hypoxanthine, inosine, 4-tert-butylbenzoic acid, sorbic acid, 4-methoxybenzoic acid,

4-hydroxybenzoic acid, 4-bromobenzene carboxylic acid, 3,4-dihydroxybenzoic acid,

cinnamic acid, diphenylamine, caffeine, theobromine, theophylline and quercetin are

from Aladdin Reagent Ltd., Shanghai, China, and diltiazem hydrochloride, cefradine,

piperacillin sodium, cefamandole sodium, cefuroxime sodium, cefotaxime sodium

and ceftezole sodium, cephradine, biapenem, doripenem, ertapenem and meropenem

were from Zhejiang Hisoar Pharm. & Chem. Co., Ltd., Taizhou, China and filtered

with a 0.45-mm membrane filter (organic membrane, BJ225-PT, Shanghai Jiapeng

Science and Technology Ltd., Shanghai).

Instruments

FT-IR measurements were performed on a Nicolet 5SXC (USA). 1H NMR was

carried out on a Bruker 400 (Germany). Elemental analysis was measured on an

elemental Vario EL III (Germany); High resolution mass spectra (HRMS) were

performed using a Bruker Daltronics MicroTof.

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Procedure:

i)N

N S

O

O

N3 HCl

ii) CuSO4 5H2O , K2CO3MeOH,r.t

O

O

Si

OEt HN

HN

OSiO2

N

NN

O

O

Si

OEtHN

HN

O

SiO2

O2N Raney-Ni/H2

+

Cu(I), MeOH:H2O=1:240 oC, 42h

5

1 23

4

OH

OHN

H2N

OH

OHN

N3

OH

OHN

3

OH

OH

N

Fig. S1 preparation path for the click N,N-dimethylamino-1,3-propanediol

Step 1. Preparation of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol 3

Step 1, To a solution of 1-(p-nitrophenyl)-2-N,N-dimethylamino-1,3-propanediol

1(10g, 42mmol ) in 100mL methanol, 1g of Raney Ni was added, these suspension

connected with a hydrogen balloon was stirred at room temperature for 10h. After the

substrate was consumed(detected by TLC), the suspension was filtered to remove

solid material and the organic layer was concentrated to left 8.3g of 2, yellow solid,

yield 95%, which was pure enough for the next reaction. 1H NMR (400 MHz, CDCl3):

δ 7.13 (d, 2 H, J = 8.4 Hz), 6.64 (d, 2 H, J = 8.4 Hz), 4.31 (d, 1 H, J = 10.0 Hz),

3.50-3.46 (m, 1 H), 3.38-3.34 (m, 1 H), 2.70-2.64 (m, 1 H), 2.50 (s, 6 H), 1.25 (s, 2 H)

Step 2. To a mixture of 2 (6.3g, 30 mmol ) , CuSO4.5H2O (80mg, 0.32mmol), K2CO3

(12.4g, 90mmol) in 90mL methanol was stirred on an ice-water bath, and

imidazole-1-sulfonyl azide hydrochloride(9.4g, 45mmol, prepared by literature

method) was added by several portions.The mixture was stirred at room temperature

for 10h. After all of 2 was consumed, the suspension was filtered and remove all

volatiles to give a residue, which was purified by silica gel column to get 5.6g brown

oil 3, yield 79%.1H NMR (400 MHz, CDCl3): δ 7.33 (d, 2 H, J = 7.2 Hz), 6.98 (d, 2 H,

J = 7.2 Hz), 4.38 (d, 2 H, J = 9.6 Hz), 3.52-3.47 (m, 1 H), 3.42-3.39 (m, 1 H),

2.64-2.59 (m, 1 H), 2.50 (s, 6 H); 13C NMR (100MHz, CDCl3): δ 139.7, 138.8, 128.6,

119.2, 71.4, 70.7, 58.2, 41.5; IR (KBr cm-1): 2100 (-N3-), 3350 (-OH-), HRMS-ESI

Calcd for C11H17N4O2 [M+H] + 237.1352, found: [M+H]+ 237.1346.

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Step 3. Immobilization of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol on

alkynes’ modified silica beads

0.44g sodium ascorbate in 5mL water was added to a solution of CuSO4.5H2O (0.28g

in 5mL H2O), then alkyne-modified silca beads (5g ) and 3 (5g in 80mL methanol)

were added successively. The mixture was stirred slowly at room temperature for 48 h

then filtered. The filter cake was washed with MeOH (300 mL), 10% EDTA (500 mL),

water (50oC, 300 mL), MeOH (200 mL), THF (200 mL), MeCN (200 mL) and

acetone (200 mL) in turn. The solid material was collected and vacuum dried at 50oC

for 10h. IR (KBr cm-1): 3400 (-OH-).

Fig. S2 the natural logarithmic plots of retention factors lnk versus water content ψ in mobile

phase. Chromatographic conditions: flow rate 1mL min-1; 35oC; mobile phase:

acetonitrile/water(v/v) and water changed from 10-30% (v); UV: 254 nm. Model

sample: uracil, 5-methyluridine, adenine, cytosine, hypoxanthine and inosine

Correlation coefficients were calculated by the following formula:

∑ ∑ ∑∑

∑ ∑ ∑

−−

−=

2222 )ln(ln)ln(ln

lnlnlnln

ψψ

ψψ

nkkn

kknR

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Table S1 Elemental analysis of alkyne-silica and click–DMAPD stationary phase

Stationary phase C% N% Surface coverage (μmol m−2)

Alkyne-silica 8.53 2.54 3.88

Click-DMAPD 15.38 5.06 2.82

Table S2 Retention factors of nucleosides and bases at different water content a

Compounds Retention factors at different acetonitrile content (v %)

90 80 70 60 50 40 30 20 10

uracil 1.62 0.99 0.69 0.55 0.48 0.45 0.46 0.51 0.60

5-methyluridine 3.08 1.35 0.78 0.58 0.47 0.42 0.42 1.09 0.59

adenine 4.09 1.83 1.17 0.83 0.69 0.64 0.69 0.89 1.41

cytosine 5.35 2.13 1.17 0.68 0.49 0.39 0.36 0.37 0.44

hypoxanthine 5.95 2.53 1.48 0.99 0.80 0.73 0.74 0.88 1.22

inosine 10.63 3.45 1.68 1.01 0.74 0.63 0.61 0.70 0.94

a Column temperature: 35 oC. Flow rate: 1.0 mL min-1. UV detection: 254 nm, t0=1.6

min.( determined by using toluene as analyte)

Table S3 Retention retention factors of model compounds at different buffer pH valuesa

Compounds Retention factors at different buffer pH values

6.0 4.5 3.3

uracil 1.26 1.27 1.30

5-methyluridine 2.03 2.06 2.13

Adenine 2.63 2.62 2.64

Cytosine 3.36 3.45 3.44

hypoxanthine 3.56 3.45 3.44

Inosine 5.64 5.55 5.62

4-hydroxybenzoic acid -- -- 4.61

4-tert-butylbenzoic acid -- -- 1.36

theophylline 1.03 0.96 0.89

a Mobile phase: ACN/10 mM ammonium formate (85/15, v/v) . Column temperature: 35 oC.

Flow rate: 1.0 mL min-1. UV detection: 254 nm, t0=1.6 min. (determined by using toluene as

analyte)

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Table S4 Retention factors of model compounds at different ammonium formate

concentrationsa

Compounds Retention factors at different ammonium formate concentrations

10 mM 20 mM 40 mM

uracil 1.30 1.31 1.33

5-methyluridine 2.13 2.14 2.18

adenine 2.64 2.64 2.66

cytosine 3.44 3.46 3.51

hypoxanthine 3.44 3.66 3.83

Inosine 5.62 5.68 5.79

4-hydroxybenzoic acid 4.61 2.92 1.89

4-tert-butylbenzoic acid 1.30 1.31 1.33

theophylline 2.13 2.14 2.18

aMobile phase: ACN/ammonium formate, pH=3.3, (85/15, v/v) for model compounds. Column

temperature: 35 oC. Flow rate: 1.0 mLmin-1. UV detection: 254 nm, t0 = 1.6 min. (determined

by using toluene as analyte)

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Fig. S3 the isocratic separation of seven organic acids on click-DMAPD (a, 5 µm, 150

mm×4.6 mm i.d.), Chromatography conditions: 85% ACN/15% aqueous ammonium

formate (20mM, pH= 3.3, v/v), the column temperature: 35oC. Flow rate: 1mLmin-1.

Wavelength: 254 nm, and Atlantis HILIC-silica (b, 5µm, 150 mm×4.6 mm i.d.), Chromatography conditions: 95% ACN/5% aqueous ammonium formate (50mM,

pH= 3.2, v/v), the column temperature: 35oC. Flow rate: 1mLmin-1. Wavelength: 254

nm. Sample: the mixed acids. Injection volume: 5μL. Peak identification: (1)

4-tert-butylbenzoic acid, (2) sorbic acid, (3) 4-methoxybenzoic acid, (4) cinnamic acid,

(5) 4-hydroxybenzoic acid, (6) 4-bromobenzene carboxylic acid, (7)

3,4-dihydroxybenzoic acid. The relative mass ratios of these probe compounds is (1):

(2): (3): (4): (5): (6): (7) = 8: 1: 2: 3: 3: 13: 12, and the injection of sorbic acid (2) was

calculated to be 0.234μg

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Fig. S4 the isocratic separation of five organic bases on click-DMAPD (a, 5 µm, 150 mm×4.6

mm i.d.) and Atlantis HILIC-silica (b, 5µm, 150 mm×4.6 mm i.d.). Chromatography conditions: 95% ACN/5% H2O (v/v), the column temperature: 35oC. Flow rate:

1mLmin-1. Wavelength: 254 nm. Sample: the mixed bases. Injection volume: 5μL.

Peak identification: (1) diphenylamine, (2) caffeine, (3) theobromine, (4) theophylline,

(5) quercetin. The relative mass ratios of these probe compounds is (1): (2): (3): (4):

(5) = 1: 2.1: 1.5: 1.6: 3, and the injection of diphenylamine (1) was calculated to be

0.176μg

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Fig. S5 Isocratic separation of seven cephalosporins on click-DMAPD (a, 5µm, 150 mm × 4.6 mm i.d.), chromatography conditions: mobile phase, ACN/20 mM ammonium

formate(pH, 3.2) (60/40, v/v). Atlantis HILIC-silica (b, 5µm, 150 mm×4.6 mm i.d.). chromatography conditions: mobile phase A, ACN, mobile phase B, 10mM aqueous

ammonium formate (pH=3.4), . 0-8min., 90% A, 8-13min., 90%-80% A,

13-40min., 80% A. The column temperature: 35 oC. Flow rate: 1 mLmin-1. Inject

volume, 3 μL. Wavelength: 254 nm. Peak identification: (1) diltiazem hydrochloride,

(2) cefradine, (3) piperacillin sodium, (4) cefamandole sodium, (5) cefuroxime

sodium, (6) impurity in cefamandole sodium, (7) cefotaxime sodium, (8) ceftezole

sodium. The relative mass ratios of these probe compounds is (1): (2): (3): (4): (5): (7):

(8) = 2.2: 1.1: 2.1: 1.3: 1: 1.2: 1.9, and the injection of cefuroxime sodium (5) was

calculated to be 4.800μg

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Fig. S6 Gradient separation of four carbapenems on click–DMAPD (a, 5µm, 150 mm × 4.6 mm i.d.)

and Atlantis HILIC-silica (b, 5µm, 150 mm×4.6 mm i.d.). Chromatography conditions: mobile phase A, ACN, mobile phase B, 20mM aqueous ammonium formate, pH=3.1.

Gradient gradient eluent: 0–10 min, 75% A, 10–20 min, 75–40% A, 20–30 min, 40% A. The

column temperature: 35oC. Flow rate: 1 mLmin-1. Inject volume, 3 μL. Wavelength: 254 nm.

Peak identification: (1) biapenem, (2) meropenem, (3) doripenem and (4) ertapenem. The

relative mass ratios of these probe compounds is (1): (2): (3): (4) = 1: 2: 2: 2.4, and the

injection of biapenem (1) was calculated to be 5.000μg

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Fig. S7 Retention stability of six nucleosides and bases on click-DMAPD by continuously

injecting fifteen times of the mixed nucleosides and bases. Mobile phase:

acetonitrile/H2O (85/15, v/v). The column temperature: 35 oC. Flow rate: 1mLmin-1.

Inject volume, 1 μL. Wavelength: 254 nm. Peak identification: (1) uracil, (2)

5-methyluridine, (3) adenine, (4) cytosine, (5) hypoxanthine, (6) inosine

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Parts of spectrums

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Fig. S8 1H NMR spectrums for related intermediates

Fig. S9 IR spectrum of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol

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Fig S10 Mass of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol

Fig. S11 IR spectrum of Alkyne-silica

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Fig. S 12 IR spectrum of Click-DMAPD

HN

HN

O O

Uracil 5-methyluridine

N

NN

NH

NH2

adenine

NH

N

OH2N

cytosine hypoxanthine

HN

NN

N

OH

HO

HO

HO

O

N

OHN O

HO

HO

HO

O

N

NO

NH

N

Inosine

Fig. S 13 Structures of nucleoside and bases for investigation

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cefotaxime sodium

S

N

NaO O

O

HNH

O

N

O

O

MeO

N

S

NH2

ceftezole sodium

S

N

NaO O

O

HNH N

O

S

N

S

N

NN

N

diltiazem hydrochloride

N

S

N

O

O

OMe

O

cefradine

S

N

HO O

O

HNH

O

H2N

N

S

O

ONaO

HHN

O

NH

N

O

N O

O

piperacillin sodium

cefamandole sodium

S

N

NaO O

O

HNH

O

HO

S

NN

N

N

cefuroxime sodium

S

N

NaO O

O

HNH

O

N

O

O

H2N

MeO

O

Fig. S14 Structures of seven cephalosporins for investigation

N

O

HO H H

O

S

N

N

N

O

1 biapenem

N

O

HO H H

HO

S

HN

O

NH

3 doripenem

S

NH2

O

O

4 ertapenem

N

O

HO H H

HO

S

HN

O

NH

O

HO2CN

O

HO H H

HO

S

HN

O

2 meropenem

N

O

Fig. S15 Structures of four carbapenems for investigation