Biomedical Engineering Reference
In-Depth Information
Hyphenating liquid chromatography (with its inherent limited separation power)
with simple low resolution molecular mass-selective detection devices as single stage
quadrupole or ion trap MS instruments (LC-MS) was soon quickly recognized to
offer only limited selectivity when used for quantitative target analyses of complex
biological samples. High resolution instruments using time-of-flight (ToF) detection
units did prove to be highly selective, but showed limited linear ranges; a prerequisite
for most quantitative assays. The introduction of tandem mass spectrometry (MS/
MS), which involves the coupling of two quadrupole mass filters with an interposed
collision cell, initially seemed to overcome these limitations because the fragmenta-
tion pattern of target analytes became incorporated into analyte detection.
Summarizing, the essential strengths of LC-MS/MS technology for laboratory
[
1
] medicine can be listed as:
1 .
Speci fi city
. The potentially very high analytical specificity of tandem mass spec-
trometry as HPLC detector results from using the molecular mass of the analyte
and its specific disintegration behaviour as detection principle.
2 .
Wide range of applicability with good practicability
. In contrast to GC-MS as the
“classical” mass spectrometry technique, the application of LC-MS/MS is not
limited to volatile molecules (usually with molecular weights below 500 Da).
Furthermore, aside from highly polar analytes (i.e. amino acids), sample prepa-
ration is usually simple and does not include derivatization techniques. Mass
spectrometry detected LC assays are generally optimized to shorter runtimes.
Hence, compared to GC-MS, far higher sample throughput can be realized.
3 .
Flexibility
. New assays can typically be developed in-house with a high degree
of flexibility and within a short time.
4 .
Information rich detection
. A large number of quantitative or qualitative results
can be obtained from a single analytical HPLC-MS/MS run, since due to the fast
ion selection electronics, multiparametric, quasi parallel analyses can be per-
formed with a mass spectrometer.
At present no MS manufacturer can offer complete solutions that meet the high
analytical demands of a routine laboratory performing LC-MS/MS analytes. None
of the mass spectrometers, let alone the complex LC-MS/MS instrument combina-
tion, is certified according to the in vitro diagnostic directive (IVDD) 98/79/EC of
the European Community (“CE certified”) or FDA approved. Generally there is no
bidirectional connection of the MS control and evaluation software to the laborato-
ry's electronic data processing. The use of self-generated scripts for the transmis-
sion of work lists to the MS or for retransmission of the measured values continues
to be common practice. With few exceptions (IVD-CE certified assays by some
providers) the manufacture of consumables (mobile solvents, precipitants) is still in
the hands of the local laboratory.
Accordingly it is costly to establish and validate an “in house” LC-MS/MS instal-
lation that should track the prevailing recommendations and guidelines of interna-
tional forums, such as CLSI (
www.clsi.org
), FDA (
www.fda.gov
) EMEA (
www.
ema.europa.eu
), or ICH (
www.ich.org
). When selecting equipment (design quali fi -
cation, DQ) and furnishing the workplace (installation qualification, IQ) it is necessary
