Transcript Solid Phase Extraction (SPE) Method Development Made Easier

Solid Phase Extraction (SPE) Method Development Made Easier
aLesego
Mmualefe, bCarol H. Ball, bDennis Blevins and bStephen Smith Jr.
aChemistry Dept., University of Botswana, Private Bag UB00704, Gaborone, Botswana . bAgilent Technologies, 2850 Centerville Rd., Wilmington DE 19808
Introduction
20
B
A
C
0
SAX Spiked buffer,
Formic acid/MeOH
fraction
300
4
≤15
≤30
min
6
5
5
4
Number of
3
compounds
2
4
Number of
3
compounds
2
MAX
The load, wash, MeOH fraction, and each of 3 x formic acid/MeOH fractions after MAX
and SAX clean-up of saline phosphate buffer spiked at 20 – 120 µg/ml.
 The fractions were dried and reconstituted in NaH2PO4 (25 mM, pH 2.5) and100
injected
90
Comparison of reconstitution solvents
MAX
SAX
SAX
1
0
≤5
≤15
Elution tim e (m in)
Ibu.
≤30
Loss of analytes through the different SPE steps
100
90
2% formic acid in MeOH
6
>30
10
≤5
>30
≤15
≤30
>30
% Recovery
≤5
8
 Interference of Vit B3 peak due to formic acid/MeOH matrix
C
NH4COOH (0.1M, pH2.0)/ACN [10:90 v/v]
0
6
 Vit B3 eluting with the MeOH peak?
B
0
Nap.
 Drying gave less than 10% differences in recoveries for neutral compounds.
 Recoveries of acidic compounds reduced by more than 40% after drying.
 The sorbents were not dried in further experiments
 Note
 NH4COOH (0.1 M, pH 4.0) / ACN [10:90 v/v]
 NH4COOH (0.1 M, pH 2.0) / ACN [10:90 v/v]
 Formic acid / MeOH [2:98 v/v]
1
Keto.
Figure 6: Comparison of % recoveries on after drying MAX & SAX
sorbents during SPE
Figure 3: Chromatogram of test compounds after injection of SPE eluates
Injected individual compounds and eluted with different solvent systems
SAX
Sali.
Acidic compounds
SAX Blank, MeOH
fraction
 Optimization of elution conditions
1
VitB3
0
Experimental
MAX
Nortri.
SAX Blank, formic
acid/MeOH fraction
2
4
Num ber of
3
com pounds
2
Seco.
SAX Spiked buffer,
MeOH fraction
100
 Make a comparison with a competitor product
5
40
400
 To evaluate a packed column approach to predict optimum experimental conditions for
SPE
6
80
60
SAX
100
90
80
70
60
50
40
30
20
10
0
100
Injection of separate eluates
200
 To evaluate a new ion exchange sorbent for the clean-up of neutral and acidic
pharmaceutical compounds
A
MAX
SAX
Neutral com pounds
Objectives
NH4COOOH (0.1M, pH4.0)/ACN [10:90 v/v]
MAX
% Recovery

Condition: 1 ml MeOH.
Equilibrate: 1 ml H2O.
Load: 1 ml phosphate saline buffer (pH 7) spiked with 20 – 120 µg/ml std mix.
Wash: 1 ml Sodium acetate (50 mM, pH 7, 5% MeOH).
Elute A: 1 ml MeOH (neutral & basic compounds).
Elute B: 1 ml formic acid / MeOH [2:98] (acid compounds).
% Recovery
This poster presents a “column-based” approach to method development on a new
ion-exchange SPE sorbent material. Optimisation of the conditioning, loading, washing
and elution steps of the SPE procedure were achieved on an HPLC column packed
with SPE sorbent employing a standard quaternary HPLC pumping system. The
optimised method was then transferred to the SPE cartridge format where recoveries
were calculated and parameters such as drying were evaluated.
SPE protocol
1.
2.
3.
4.
5.
6.
Solid phase extraction (SPE) is one of the simplest yet most effective and versatile
methods of sample preparation. It is used for both clean-up and pre-concentration of
analytes in various matrices. Unlike its predecessor, liquid-liquid extraction (LLE), it
employs minimal organic solvents (usually a maximum of 10 ml as compared to the
typical 100 ml volumes used in LLE). SPE also offers shorter processing times as well
as being cost effective. SPE has found applications in the food, environmental and
pharmaceutical industries due to the different chemistries offered by the silica,
alumina, florisil and polymeric based phases available. SPE offers different separation
modes such as reversed phase, normal and ion exchange depending on the sorbent
type.

Effect of drying
Transfer of method to SPE cartridges

#301 HPLC2008
Baltimore, MD
USA.
Elution time (min)
Elution time (min)
Figure 2: Graphs A, B & C showing the numbers of compounds eluted during
different time frames when using different elution solvents. MAX refers to OASIS
MAX sorbent while SAX refers to experimental SAX sorbent.
80
80
70
70
E2-3
30
20
E1
E2-1
50
E1
40
W
30
E2-2
L
20
E2-3
10
0
Std mix in saline
phosphate buffer (pH
7.0
W
60
E2-2
60
50
40
L
E2-1
10
Seco. (MAX)
 Formic acid / MeOH [2:98 v/v] eluted most compounds in the void volume
Seco. (SAX)
Nortri. (MAX)
0
Nortri. (SAX)
VitB3
(M AX)
VitB3
(SAX)
Sali.
(M AX)
Sali
(SAX)
Keto.
(M AX)
Keto.
(SAX)
Nap.
(M AX)
Nap.
(SAX)
Ibu.
(M AX)
Ibu.
(SAX)
Figure 7: Cumulative graphs showing the presence of the analytes in the different
fractions during SPE (N=3) where L=load; W=wash; E1=MeOH fraction; E2-1, E2-2 &
E2-3=1st, 2nd & 3rd acidic fractions.
Std mix in NaH2PO4 (25
mM, pH 2.5
 Note:
 Both MAX & SAX sorbents gave ~80% recoveries for the neutral compounds in the
MeOH fraction.
 Almost 50% of Vit B3 is eluted in the MeOH fraction (E1) on MAX sorbent while 20% is
lost in the same fraction on SAX
Binding & washing conditions
 Injected individual compounds in phosphate saline buffer (pH 7.0)
Std mix in formic acid
/MeOH [2:98 v/v]
 No breakthrough observed
 More than 50% of salicylic acid is recovered in the 2nd acidic fraction
 Flushed column with sodium acetate (50 mM, pH 7.0)
Std mix in MeOH
 No breakthrough observed
Reproducibility studies
0
0
Separation of test compounds
2
4
6
8
10
min
Figure 4: Chromatograms of std mixtures (20 – 120 µg/ml) dried and re-
Table 1: Reproducibility studies (N=6) & spiking at at 20 –
120 µg/ml spiking level
constituted in different solvents
Vit B3
•
 Agilent 1200 HPLC
Not recovered in organic phase containing reconstitution solvents
MAX
 Vit B3 eluted in the solvent front
 Autosampler, quaternary / binary pumps, temperature controlled column
compartment, diode array detector at 222, 230, 242 and 258 nm.
 Column: Eclipse Plus C18 column (4.6 x 50 mm x 3.5 µm)
 Mobile phase composition:

Solvent A = NaH2PO4 (25 mM, pH 2.5)

Solvent B = MeOH
1.
Vit B3………..258 nm
2.
Salicylic acid..230 nm
3.
Secobarbital...222 nm
4.
Nortriptyline….242 nm
5.
Ketoprofen…..258 nm
6.
Naproxen…….230 nm
7.
Ibuprofen……..222 nm
2
Secobarbital
Nortriptyline
VitB3
Salicylic acid
Ketoprofen
Naproxen
Ibuprofen
 chosen for re-constituting dried eluates
Comparison of SPE recoveries
76
81
45
11
92
89
88
% RSDs %Recovery (N=6)
2.7
91
1.8
94
2.2
71
8.9
98
2.0
97
1.8
98
1.5
98
% RSDs
1.3
1.0
0.9
0.7
1.0
0.6
1.3
 % recoveries were calculated and compared
%B
1
50
60
80
1
Conclusions
 An SPE method was successfully developed “on-column” and the optimized
conditions transferred to cartridge format.
 Effect of drying the sorbent during the SPE procedure
7
6
3
NaH2PO4 (25mM, pH2.5) gave a higher peak area for secobarbital
 MAX and SAX sorbents were dried under vacuum for 10 min after the conditioning
step.
Gradient elution
t (min)
0-1
2
5-6
7-8
10
•
%Recovery (N=6)
SAX
 Slight positive effect on the neutral compounds
 Lower recoveries for the acidic compounds
 drying not recommended for SAX sorbent.
4
5
1
Further work
 Clean-up of real samples such as blood serum, sewage water etc.
Acknowledgements
 MAX sorbent
 Recoveries 11 – 92%
 %RSDs 1.5 – 8.9
0
1
2
3
4
5
6
7
8
 SAX sorbent
min
Figure 2: Separation of neutral and acidic pharmaceutical compounds on an
Eclipse Plus C18 4.6 x 50 mm x 3.5 µm.
Figure 5: Comparison of % recoveries after MAX & SAX SPE of
phosphate saline buffer spiked at 20 – 120 µg/ml.
 Recoveries 71 – 98%
 %RSDs 0.6 – 1.3
 performed better than MAX.
 Agilent Technologies Inc.
 CSD team members
 University of Botswana
 Department of Chemistry