1960 – mid 1970’s Clean-up of biological specimens, such as whole blood, serum, plasma and urine, for clinical chemical analysis does not change significantly between 1960 and late 1970’s. Pretreatment is centrifugation and liquid-liquid extraction (LLE) prior to GC-MS analysis. Clinical chemical analysis is highly selective, with enzyme catalyzed and/or antibody triggered/boosted chemical reactions for quantitation of small molecules, such as metabolites and endogenous compounds, and large molecules like functional proteins. Late 1970s - 1999 HPLC and a broad spectrum of stationary phases focus attention on sample pretreatment. In subsequent years, solid phase extraction (SPE) becomes the dominant clean-up principle for pretreatment of body fluids, especially for HPLC-UV/VIS, fluorescence detector (FD) or electrochemical detector (ECD) analysis of small molecules in therapeutic drug monitoring (TDM), and for forensic specimens. Routine SPE is mainly performed manually or semi-automatically by dedicated liquid/SPE handling systems.
1960 – mid 1970’s
Clean-up of biological specimens, such as whole blood, serum, plasma and urine, for clinical chemical analysis does not change significantly between 1960 and late 1970’s.
Pretreatment is centrifugation and liquid-liquid extraction (LLE) prior to GC-MS analysis.
Clinical chemical analysis is highly selective, with enzyme catalyzed and/or antibody triggered/boosted chemical reactions for quantitation of small molecules, such as metabolites and endogenous compounds, and large molecules like functional proteins.
Late 1970s - 1999
HPLC and a broad spectrum of stationary phases focus attention on sample pretreatment.
In subsequent years, solid phase extraction (SPE) becomes the dominant clean-up principle for pretreatment of body fluids, especially for HPLC-UV/VIS, fluorescence detector (FD) or electrochemical detector (ECD) analysis of small molecules in therapeutic drug monitoring (TDM), and for forensic specimens.
Routine SPE is mainly performed manually or semi-automatically by dedicated liquid/SPE handling systems.
~2000
HPLC instruments and software integrate SPE-based sample clean-up: SPE-LC is born.
On-line SPE-LC relies on tailor-made packings using restricted-access materials in small SPE-columns (20 x 2 mm inside diameter).
SPE-LC becomes increasingly attractive, saving costs on consumables and salaries, and allows complete automation. Sample throughput is a rate-limiting step.
Mid-2000s
Tandem mass spectrometry (MS/MS) enters routine clinical-chemistry laboratories with the message “dilute and shoot”.
It becomes apparent that when operating MS/MS in electrospray ionization (ESI)-mode, ionization might be decreased or enhanced – so-called “matrix effects”.
SPE packings to remove phospholipids and multidimensional SPE are introduced to maximize sample clean-up and minimize matrix effects.
2008~
Dried blood spot (DBS) and dried spots of other biofluids, such as urine and plasma, become popular, especially for Phase I studies in drug development (See “Pharma's DBS Dilemma”, page 36).
The combination of (micro) sampling, ease of shipment and storage using a single filter card boost its popularity.
2013 and beyond...
SPE
Intra-laboratory turn-around times in laboratory medicine are 3 min to 3 hours for common analytes. To compete, SPE is being miniaturized, multiplexed and/or hyphenated with UPLC under normal or high linear flow (TurboFlow).
MS
Besides well-established GC-MS systems, routine application of (UP)LC-MS/MS, TOF-MS, DART-MS and other MS platforms in the clinical setting is slowly but steadily catching on.
To match classical clinical-chemical analyzers, improvements in ease of operation, robustness, downtime, and 24/7 runtime are required.
DBS
DBS has potential use in outpatient thereapeutic drug monitoring (see “Spot On”, theanalyticalscientist.com/issues/0413/401).
Cell-Disintegrated Blood (CDB)
Offers the potential to simplify and fully automate the analysis of whole blood prior to SPE-LC or point-of-care-testing (POCT). POCT uses sensor technologies that involve little to no sample preparation.
Improvements in microchip and nanoscale technology will yield automated, algorithm-dependent in-situ diagnostics that obviate the need for sample preparation.
NMR
Routine application of high resolution NMR in the classification of subclasses of low-density-lipoproteins has been a door opener in laboratory medicine.
NMR, in principle, does not require sample clean-up, yet allows quantitative analysis of highly complex bodily fluids, such as urine.
NMR-based analysis protocols will become attractive in systems biology-based profiling and, eventually, in personalized (laboratory) medicine.
Karl-Siegfried Boos and Rosa Morello are at the Laboratory of BioSeparation, Institute of Clinical Chemistry, Medical Center of the University of Munich, Germany.
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