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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
2
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.
3
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.
4
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
5
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)
6
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)
7
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
8
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
9
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
10
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
11
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
12
Parts of spectrums
13
Fig. S8 1H NMR spectrums for related intermediates
Fig. S9 IR spectrum of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol
14
Fig S10 Mass of 1-(p-azidophenyl)-2-N,N-dimethylamino-1,3-propanediol
Fig. S11 IR spectrum of Alkyne-silica
15
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
16
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