Method development on-line SPE-LC



LevelBasic

In the next paragraphs we explain the operational procedure and steps to develop a method for on-line SPE-LC of biofluids.

In addition a number of examples are given.

Step 1a

Step 1a:  

  • Fractionation of biofluid(s) into analyte(s) and sample matrix   (= Adsorptive extraction and enrichment of analyte(s))   
  • Determine complete elution of sample matrix =  tM [min]   
  • Calculate valve switching time =  tV1 [min]    (= End of fractionation step and coupling of SPE-column with LC-column )

Determine tM

Instrumental set-up for the determination of tM.

Instrumental Set-up: Determination of tMInstrumental Set-up:  Determination of tM

Procedure :
1. Couple SPE-column directly to an UV detector set at 280 nm
2. Inject biofluid (e.g. plasma) and record elution profile of sample matrix

Sample Fractionation Step:   
  • Use a mobile phase compatible with the sample:
    • amount of organic modifier
    • pH
  • Use an adequate flow rate for loading the SPE-column with the sample

Example:  Determination of tM:  Human Plasma

Determination of tM: Human Plasma Determination of tM:  Human Plasma

Step 1b

  • Fractionation of biofluid(s) into analyte(s) and sample matrix 
  • Then determine breakthrough of analyte(s) = tA  [min]

Examples of compounds (Antimycotics / Antifungals) that we use as example in this tutorial:

Antimycotics / AntifungalsAntimycotics / Antifungals

Determine tA

Instrumental set up to determine tAInstrumental set up to determine tA

Procedure:

  1. Couple SPE-column to appropriate detector
  2. Use mobile phase composition and flow-rate as for tM
  3. Inject analyte(s) and record elution profile    Important: Conditio sine qua non :   tA >  tM

Example:  the determination of tA:  Antimycotic (Voriconazole)

Determination of tA: Antimycotic (Voriconazole)Determination of tA:  Antimycotic (Voriconazole)

Step 2.

Step 2 :  

  • Transfer of analyte(s) from SPE-column to analytical    HPLC-column     
    • Determine complete elution of analyte(s) = tT [min]   
    • Calculate valve switching time  =  tV2 [min]    = End of transfer step and decoupling of SPE-column from LC-column

Instrumental Set-up :  Determination of tT  

Instrumental Set-up: Determination of tTInstrumental Set-up:  Determination of tT

Procedure:

  1. Load analyte(s) onto SPE-column (mobile phase I)
  2. Switch valve
  3. Use mobile phase II
  4. Desorb analyte(s) and record elution profile with a directly coupled detector

Example of the determination of tT:   Antimycotic  (Itraconazole)

Determination of tT: Antimycotic (Itraconazole)Determination of tT: Antimycotic (Itraconazole)

Calculation of valve switching times

tV1  =  ½ (tM  + tA )  =  ½ (1 + 3)  =  2 min

tV2  = tV1 +  tT  +  1  =  5 min

Calculation of valve switching timesCalculation of valve switching times

Examples

On-line SPE-LC-UV of Antimycotics: Separation of a Standard MixtureOn-line SPE-LC-UV of Antimycotics: Separation of a Standard Mixture

   On-line SPE-LC-UV of Antimycotics: Memory Effect (Carry Over)On-line SPE-LC-UV of Antimycotics: Memory Effect (Carry Over)

Memory Effect and Countermeasure: Extension of tTMemory Effect and Countermeasure:  Extension of tT

Memory Effect: Additional wash of SPE-columnMemory Effect: Additional wash of SPE-column 

On-line SPE(RP/SEC)-LC-UV Analysis of Antimycotics in Human Plasma: Final ProtocolOn-line SPE(RP/SEC)-LC-UV Analysis of Antimycotics in Human Plasma: Final Protocol

On-line SPE-LC-UV Analysis of Antimycotics: Spiked Human PlasmaOn-line SPE-LC-UV Analysis of Antimycotics: Spiked Human Plasma

On-line SPE-LC-UV Analysis of Antimycotics: Patient PlasmaOn-line SPE-LC-UV Analysis of Antimycotics: Patient PlasmaOn-line SPE-LC-UV Analysis of Antimycotics: Patient PlasmaOn-line SPE-LC-UV Analysis of Antimycotics: Patient Plasma

 Therapeutic Drug Monitoring of Antimycotics: Method Comparison On-line SPE-LC-UV versus Offline Precipitation and LC-MS/MSTherapeutic Drug Monitoring of Antimycotics:  Method Comparison On-line SPE-LC-UV versus Offline Precipitation and LC-MS/MS

Optimization

Goals

  • Maximizing Analyte enrichment SPE column lifetime
  • Minimizing Retention of interfering compounds „Matrix Effects“

Variables: 

  • SPE column packing material
  • Mobile phase composition
    • pH 
    • Additives (ion-pair reagent, org. modifier)
  • Volume
    • Sample 
    • Fractionation / Transfer step
  • SPE column size

Optimization of the transfer step

  • Goal Peak compression (in-line enrichment)
  • Approach
    • Mobile phase composition
    • Post-column dilution
    • Thermo-Desorption

On-line SPE-LC Quo Vadis?

SPE-LC will have two developments, one in the direction of miniaturization, the other is downsizing. We will describe both:

Miniaturization  

Advantages:

  • Decreased diffusion path length improves separation efficiency
  • Reduced consumption of sample (human, animal) and mobile phases
  • Diminished production of chemical and biohazardous waste
  • Significant reduction of analysis time
  • Increased throughput
  • Multiple analyses from a single sample
  • Increased sensitivity of concentration dependent detection modes e.g. UV or ESI-MS

Miniaturization of LC Techniques :  Terminology
  Column ID   Flow-rate
  conventional LC   3.2 – 4.6 mm   0.5 – 2.0 mL/min
  microbore LC   1.5 – 3.2 mm   100 – 500 µL/min
  micro LC   0.5 – 1.5 mm   10 – 100 µL/min
  capillary LC   150 – 500 µm   1 – 10 µL/min
  nano LC   10 – 150  µm   10 – 1000 nL/min

Source: Szumski M et al., Critical Reviews in Analytical Chemistry 32 (2002) 1-46

Downsizing

Downsizing
            \ Method 
  Column \
  conventional   microbore
  SPE-column   20 x 4 mm ID   20 x 1 mm ID
  Analytical column   150 x 4.6 mm ID   50 x 2.1 mm ID

On-line SPE(RP/SEC)-LC/UV of Antimycotics: Comparison of conventional with downsized SPE and LC columns (Click for full view)On-line SPE(RP/SEC)-LC/UV of Antimycotics: Comparison of conventional with downsized SPE and LC columns (Click for full view)

On-line SPE (RP / SEC) – LC / UV of Antimycotics
Comparison of conventional with microbore SPE and LC columns

Conventional   Microbore Reduction
Overall analysis time [min]     13    5.5   2.4 fold    
Eluent consumption / analysis cycle [mL]     26    4.4 5.9 fold
Sample injection volume [µL] 2.5 fold
Response Factor (Itraconazole) 1 ng = 0.23 mAU 1 ng = 0.93 mAU 4.0 fold
ESI-MS compatibility limited (1.2 mL/min) ideal (0.4 mL/min) -

Solid Phase Micro Extraction:  SPME Fiber and HPLC Interface ...... Solid Phase Micro Extraction: SPME Fiber and HPLC InterfaceSolid Phase Micro Extraction: SPME Fiber and HPLC Interface

Courtesy of Mullett W.M.
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