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Solid Phase Extraction _SPE_ Met

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Solid Phase Extraction _SPE_ Met Powered By Docstoc
					                                                                                                                                             Solid Phase Extraction (SPE) Method Development Made Easier
                                                                                                                                                                                                      Mmualefe, bCarol H. Ball, bDennis Blevins and bStephen Smith Jr.
                                                                                                                                                                                                                                 aLesego
                                                                                                                                                            aChemistry Dept., University of Botswana, Private Bag UB00704, Gaborone, Botswana . bAgilent Technologies, 2850 Centerville Rd., Wilmington DE 19808                                                                                                                                                                                                  USA.                        #301 HPLC2008
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Baltimore, MD
                                                          Introduction                                                                                                                                 Transfer of method to SPE cartridges                                                                                                                                                                                                                Effect of drying
                                                                                                                                                                                                                        SPE protocol
                                                                                                                                                                                                                         1.   Condition: 1 ml MeOH.                                                                                                                                                                    MAX        SAX                                                                                                        MAX         SAX
Solid phase extraction (SPE) is one of the simplest yet most effective and versatile                                                                                                                                     2.   Equilibrate: 1 ml H2O.
methods of sample preparation. It is used for both clean-up and pre-concentration of                                                                                                                                     3.   Load: 1 ml phosphate saline buffer (pH 7) spiked with 20 – 120 µg/ml std mix.
analytes in various matrices. Unlike its predecessor, liquid-liquid extraction (LLE), it
                                                                                                                                                                                                                         4.   Wash: 1 ml Sodium acetate (50 mM, pH 7, 5% MeOH).
                                                                                                                                                                                                                                                                                                                                                                            100                                                                                                           100
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                                                                                                                                    5.   Elute A: 1 ml MeOH (neutral & basic compounds).                                                                                                                                                                                                              90
                                                                                                                                                                                                                                                                                                                                                                             80
as being cost effective. SPE has found applications in the food, environmental and                                                                                                                                       6.   Elute B: 1 ml formic acid / MeOH [2:98] (acid compounds).                                                                                                                                                                                                    80
pharmaceutical industries due to the different chemistries offered by the silica,                                                                                                                                       Injection of separate eluates




                                                                                                                                                                                                                                                                                                                                                                                                                                                                             % Recovery
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           70




                                                                                                                                                                                                                                                                                                                                                               % Recovery
alumina, florisil and polymeric based phases available. SPE offers different separation                                                                                                                                                                                                                                                                                      60
modes such as reversed phase, normal and ion exchange depending on the sorbent                                                                                                                                                                                                                                                                                                                                                                                                             60
type.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      50
                                                                                                                                                                                                                                                                                                                                                                             40
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           40
This poster presents a “column-based” approach to method development on a new
ion-exchange SPE sorbent material. Optimisation of the conditioning, loading, washing
                                                                                                                                                                                                       400
                                                                                                                                                                                                                                                                                                                                                                             20                                                                                                            30
and elution steps of the SPE procedure were achieved on an HPLC column packed                                                                                                                                                                                                                                                                                                                                                                                                              20
with SPE sorbent employing a standard quaternary HPLC pumping system. The                                                                                                                                                                                                                   B               C

optimised method was then transferred to the SPE cartridge format where recoveries
                                                                                                                                                                                                                                                               A
                                                                                                                                                                                                                                                                                                                                                                              0                                                                                                            10
                                                                                                                                                                                                                                                                                                                               SAX Spiked buffer,
were calculated and parameters such as drying were evaluated.                                                                                                                                          300
                                                                                                                                                                                                                                                                                                                               Formic acid/MeOH                                                         Seco.                                Nortri.                                        0
                                                                                                                                                                                                                                                                                                                               fraction
                                                                                                                                                                                                                                                                                                                                                                                                                   Neutral com pounds                                                                    VitB3               Sali.               Keto.                Nap.             Ibu.
                                                               Objectives                                                                                                                              200                                                                                                                    SAX Spiked buffer,
                                                                                                                                                                                                                                                                                                                              MeOH fraction
                                                                                                                                                                                                                                                                                                                                                                                                                       Figure 6: Comparison of % recoveries on after drying MAX & SAX
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        Acidic compounds

                                                                                                                                                                                                                                                                                                                                                                                                                                            sorbents during SPE
 To evaluate a new ion exchange sorbent for the clean-up of neutral and acidic                                                                                                                                                                                                                                                SAX Blank, formic
  pharmaceutical compounds                                                                                                                                                                             100
                                                                                                                                                                                                                                                                                                                               acid/MeOH fraction
                                                                                                                                                                                                                                                                                                                                                                                                                           Drying gave less than 10% differences in recoveries for neutral compounds.
 To evaluate a packed column approach to predict optimum experimental conditions for                                                                                                                                                                                                                                                                                                                                      Recoveries of acidic compounds reduced by more than 40% after drying.
                                                                                                                                                                                                                                                                                                                               SAX Blank, MeOH
  SPE                                                                                                                                                                                                                                                                                                                          fraction                                                                                    The sorbents were not dried in further experiments
 Make a comparison with a competitor product                                                                                                                                                            0


                                                                                                                                                                                                                                    2                      4                        6                           8                   10             min




                                                          Experimental                                                                                                                                       Figure 3: Chromatogram of test compounds after injection of SPE eluates
                                                                                                                                                                                                                                                                                                                                                                                               Loss of analytes through the different SPE steps
                                                                                                                                                                                                                                         Note                                                                                                                    The load, wash, MeOH fraction, and each of 3 x formic acid/MeOH fractions after MAX
            Optimization of elution conditions                                                                                                                                                                                                                                                                                                                   and SAX clean-up of saline phosphate buffer spiked at 20 – 120 µg/ml.
                               Injected individual compounds and eluted with different solvent systems                                                                                                                                      Interference of Vit B3 peak due to formic acid/MeOH matrix
                                     NH4COOH (0.1 M, pH 4.0) / ACN [10:90 v/v]                                                                                                                                                                                                                                                                                              The fractions were dried and reconstituted in NaH2PO4 (25 mM, pH 2.5) and100
                                                                                                                                                                                                                                                                                                                                                                                                                                                        injected
                                     NH4COOH (0.1 M, pH 2.0) / ACN [10:90 v/v]
                                                                                                                                                                                                                                                  Vit B3 eluting with the MeOH peak?
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          90
                                     Formic acid / MeOH [2:98 v/v]                                                                                                                                                                                                                                                                                                                            100                                                                                        80
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       L
                                                                                                          B                                                            C                                                                                                                                                                                                                        90
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          70
                              A
             NH4COOOH (0.1M, pH4.0)/ACN [10:90 v/v]
                                                                                 NH4COOH (0.1M, pH2.0)/ACN [10:90 v/v]                                       2% formic acid in MeOH
                                                                                                                                                                                                    Comparison of reconstitution solvents                                                                                                                                                       80
                                                                                                                                                                                                                                                                                                                                                                                                70
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     E2-3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          60
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       W




                                                                                                                                                                                                                                                                                                                                                                                  % Recovery
                                                                                  6                                                                    6                                                                                                                                                                                                                                                                                                             E2-2                                                                                                              E1
             6
                                                                                  5                                                                    5                                                                                                                                                                                                                                        60                                                                                        50
             5
                                                                                   4                                                                    4                                                                                                                                                                                                                                                                                                            E2-1
   Num ber of
              4
                                                                         Number of
                                                                                   3
                                                                                                                                              Number of
                                                                                                                                                        3                                                                                                                                                                                                                                       50                                                                                                                                                                                     E2-1
  com pounds
              3
              2
                                                              MAX       compounds
                                                                                   2
                                                                                                                                       MAX   compounds
                                                                                                                                                        2
                                                                                                                                                                                              MAX
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     E1                   40
                                                              SAX                                                                      SAX                                                    SAX
                                                                                                                                                                                                                                                                                                                                                                                                40
             1
             0
                                                                                  1

                                                                                  0
                                                                                                                                                       1
                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                     W                    30                                                                                           E2-2
                  ≤5         ≤15          ≤30       >30                                ≤5        ≤15          ≤30            >30
                                                                                                                                                            ≤5     ≤15          ≤30
                                                                                                                                                                   Elution time (min)
                                                                                                                                                                                        >30
                                                                                                                                                                                                                                                                                                                                                                                                30
                            Elution tim e (m in)                                                Elution time (min)
                                                                                                                                                                                                                                                                                                                                                                                                20                                                                   L                    20                                                                                           E2-3
         Figure 2: Graphs A, B & C showing the numbers of compounds eluted during                                                                                                                                                                                                                                                                                                               10                                                                                        10
                                                                                                                                                                                                                                                                                                                        Std mix in saline
                                                                                                                                                                                                                                                                                                                                                                                                 0
        different time frames when using different elution solvents. MAX refers to OASIS                                                                                                                                                                                                                                phosphate buffer (pH
                                                                                                                                                                                                                                                                                                                        7.0                                                                                                                                                                0
                                                                                                                                                                                                                                                                                                                                                                                                     Seco. (MAX)    Seco. (SAX)    Nortri. (MAX)   Nortri. (SAX)
                    MAX sorbent while SAX refers to experimental SAX sorbent.                                                                                                                                                                                                                                                                                                                                                                                                                    VitB3   VitB3     Sali.    Sali    Keto.    Keto.    Nap.     Nap.     Ibu.    Ibu.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                (M AX)   (SAX)    (M AX)   (SAX)   (M AX)   (SAX)    (M AX)   (SAX)   (M AX)   (SAX)


     Formic acid / MeOH [2:98 v/v] eluted most compounds in the void volume                                                                                                                                                                                                                                                                                                                                            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 &
                                                                                                                                                                                                                                                                                                                        Std mix in NaH2PO4 (25
                                                                                                                                                                                                                                                                                                                        mM, pH 2.5
                                                                                                                                                                                                                                                                                                                                                                                                                                               E2-3=1st, 2nd & 3rd acidic fractions.
                                                                                                                                                                                                                                                                                                                                                                       Note:
                                                                                                                                                                                                                                                                                                                                                                                Both MAX & SAX sorbents gave ~80% recoveries for the neutral compounds in the
     Binding & washing conditions
                                                                                                                                                                                                                                                                                                                                                                                 MeOH fraction.
            Injected individual compounds in phosphate saline buffer (pH 7.0)
                                                                                                                                                                                                                                                                                                                          Std mix in formic acid
                                                                                                                                                                                                                                                                                                                          /MeOH [2:98 v/v]                                      Almost 50% of Vit B3 is eluted in the MeOH fraction (E1) on MAX sorbent while 20% is
                   No breakthrough observed                                                                                                                                                                                                                                                                                                                                     lost in the same fraction on SAX

                                                                                                                                                                                                                                                                                                                                                                                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                                                                                                                                                                 0


                                                                                                                                                                                                                 0                      2                      4                        6                           8                 10             min
                                                                                                                                                                                                                                                                                                                                                                                                                                            Reproducibility studies
                                                                                                                                                                                                                 Figure 4: Chromatograms of std mixtures (20 – 120 µg/ml) dried and re-
   Separation of test compounds                                                                                                                                                                                                                 constituted in different solvents
                                                                                                                                                                                                                                                                                                                                                                                                                                   Table 1: Reproducibility studies (N=6) & spiking at at 20 –
                                                                                                                                                                                                                                                                                                                                                                                                                                                   120 µg/ml spiking level
                                                                                                                                                                                                                                            Vit B3
                                                                                                                                                                                                                                            •   Not recovered in organic phase containing reconstitution solvents                                                                                                                                                  MAX                                            SAX
         Agilent 1200 HPLC                                                                                                                                                                                                                      Vit B3 eluted in the solvent front                                                                                                                                                         %Recovery (N=6)                % RSDs %Recovery (N=6)                          % RSDs
              Autosampler, quaternary / binary pumps, temperature controlled column                                                                                                                                                        •   NaH2PO4 (25mM, pH2.5) gave a higher peak area for secobarbital
                                                                                                                                                                                                                                                                                                                                                                                                                        Secobarbital                    76                   2.7        91                                   1.3
               compartment, diode array detector at 222, 230, 242 and 258 nm.                                                                                                                                                                    chosen for re-constituting dried eluates
                                                                                                                                                                                                                                                                                                                                                                                                                        Nortriptyline                   81                   1.8        94                                   1.0
         Column: Eclipse Plus C18 column (4.6 x 50 mm x 3.5 µm)                                                                                                                                                                                                                                                                                                                                                        VitB3                           45                   2.2        71                                   0.9
         Mobile phase composition:
                      Solvent A = NaH2PO4 (25 mM, pH 2.5)
                                                                                                                                                                                                                     Comparison of SPE recoveries                                                                                                                                                                       Salicylic acid
                                                                                                                                                                                                                                                                                                                                                                                                                        Ketoprofen
                                                                                                                                                                                                                                                                                                                                                                                                                                                        11
                                                                                                                                                                                                                                                                                                                                                                                                                                                        92
                                                                                                                                                                                                                                                                                                                                                                                                                                                                             8.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                                             2.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        98
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        97
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             0.7
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             1.0
                                                                                                                                                                                                                                                                                                                                                                                                                        Naproxen                        89                   1.8        98                                   0.6
                      Solvent B = MeOH                                                                                                                                                                               MAX and SAX sorbents were dried under vacuum for 10 min after the conditioning                                                                                                                   Ibuprofen                       88                   1.5        98                                   1.3
                                                                                                       Gradient elution                                                                                                step.
                            1.            Vit B3………..258 nm
                            2.            Salicylic acid..230 nm
                                                                                                        t (min)                    %B                                                                                 % recoveries were calculated and compared
                                                                                                          0-1                       1
                            3.
                            4.
                                          Secobarbital...222 nm
                                          Nortriptyline….242 nm
                                                                                                            2
                                                                                                          5-6
                                                                                                                                   50
                                                                                                                                   60
                                                                                                                                                                                                                                                                                                                                                                                                         Conclusions                                                                                                               Further work
                                                                                                          7-8                      80
                            5.            Ketoprofen…..258 nm
                                                                                                           10                       1
                            6.            Naproxen…….230 nm
                                                                                                                                                                                                                                                                                                                                                            An SPE method was successfully developed “on-column” and the optimized                                                                        Clean-up of real samples such as blood serum, sewage water etc.
                            7.            Ibuprofen……..222 nm                                                                                                                                                                                                                                                                                                conditions transferred to cartridge format.
                                                                                                                                                  7
                                                                    2
                                                                                                                                   6                                                                                                                                                                                                                        Effect of drying the sorbent during the SPE procedure
                                                                                                    3     4                                                                                                                                                                                                                                                                   Slight positive effect on the neutral compounds


                    1
                                                                                                                         5
                                                                                                                                                                                                                                                                                                                                                                              Lower recoveries for the acidic compounds
                                                                                                                                                                                                                                                                                                                                                                                  drying not recommended for SAX sorbent.                                                                                         Acknowledgements
                                                                                                                                                                                                                                                                                                                                                            MAX sorbent
                                                                                                                                                                                                                                                                                                                                                                              Recoveries 11 – 92%
                                                                                                                                                                                                                                                                                                                                                                              %RSDs 1.5 – 8.9                                                                                                                    Agilent Technologies Inc.
                                                                                                                                                                                                                                                                                                                                                            SAX sorbent
    0                   1                       2         3              4                  5                        6                 7      8              min

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    CSD team members
                                                                                                                                                                                                                                                                                                                                                                              Recoveries 71 – 98%
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  University of Botswana
          Figure 2: Separation of neutral and acidic pharmaceutical compounds on an                                                                                                                  Figure 5: Comparison of % recoveries after MAX & SAX SPE of                                                                                                              %RSDs 0.6 – 1.3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Department of Chemistry
                                                                                                                                                                                                                                                                                                                                                                                           performed better than MAX.
                            Eclipse Plus C18 4.6 x 50 mm x 3.5 µm.                                                                                                                                         phosphate saline buffer spiked at 20 – 120 µg/ml.

				
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