Biomedical Engineering Reference
In-Depth Information
In addition to hydrocarbon chains of variable length (for reversed phase SPE),
quaternary ammonium or amino groups (for anion exchange), and sulfonic acid or
carboxyl groups (for cation exchange) were introduced for mix mode extraction with
increased extraction selectivity. The third generation of SPE stationary phases was
made from hydrophobic, yet water-wettable polymeric sorbents. Several commonly
known brand and products are silica based Isolute ® SPE (Biotage, Uppsala, Sweden)
and more recently polymer based SPE sorbent such as Oasis ® SPE (Waters, Milford,
MA, USA) and Strata™ SPE (Phenomenex, Torrance, CA, USA). These products
contain generic reversed phase SPE sorbents as well as more advanced mix mode such
as strong and weak ion exchange sorbent. All of the newer SPE sorbents were made
of water-wettable polymers which provide much wider pH range from 0 to 14.
Another advanced and novel sample preparation technique is online solid phase
extraction (online SPE). Although the extraction mechanism is as the same as tradi-
tional offline SPE, online SPE offers several advantages. Because sample prepara-
tion is carried out during analysis, it eliminates the time needed for sample
preparation thus increases throughput significantly. Additionally, because the entire
sample is eluted to the LC-MS/MS system, it may increase assay sensitivity. Finally,
because there is no manual extraction involved, it may reduce human error, potential
contamination, and inconsistent recovery.
The direct comparison of the advantage and disadvantage of offline and online
SPE was evaluated in our laboratory using raltegravir as model compound.
Raltegravir is a new class of drug used to treat HIV infection. Two separate methods
were developed to quantify raltegravir in human plasma at linear range of 1.00-
1,000 ng/mL. As shown in Table 1 , the offline extraction method was developed
using HPLC coupled with Sciex API 5000™ (MDS Sciex, Concord, Ontario) in
negative ion mode using ESI source. Strata-X 96-well plate following multiple con-
ditioning, equilibration, washing, and elution steps was used for sample prepara-
tion. A Waters Xbridge™ phenyl 2×50 mm column was utilized to achieve
appropriate retention and separation with cycle time of 3 min. In contrast, online-
SPE method uses Symbiosis™ Pharma (Spark Holland, The Netherlands) coupled
with Sciex API 4000™ in negative ion mode using ESI source. The online SPE
sample preparation is as easy as diluting plasma sample with internal standard and
buffer solution. Online SPE is conducted under high pressure with two syringe
pumps, where the HySphere™ C18HD 7 mm cartridge (Spark Holland) provides
adequate sample cleanup and chromatographic separation. The diluted sample with
ca. 3 mL of plasma is extracted and analyzed simultaneously by Symbiosis Pharma
coupled with Sciex API 4000 with a cycle time of 100 s. The analytical column was
omitted which reduces the cycle time and cost of analytical column. As shown in
Figs. 1 and 2 , because the sensitivity of online SPE is much higher than that of
offline SPE, the mass spectrometer was changed from a Sciex API 5000 to API
4000 to achieve the equivalent sensitivity at the same Lower limit of Quantitation
(LLOQ) of 1 ng/mL.
Although the advantage of online SPE is apparent, it has several limitations as
well. Because online SPE method is operated under “elute and shoot” mode, the
elution solvent has to be compatible with HPLC mobile phases. If the pH of the
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