WEBVTT

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Mass spectrometry

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allows for the identification
and quantitation of specific analytes

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with greater sensitivity and specificity
than many other lab techniques.

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Samples are brought to the lab,
sorted by test.

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The analyte of interest is then extracted
from the samples submitted to the lab,

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along with collaborators
and controls to be used on the run.

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Quantitation
of an unknown amount of analyte

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in each sample by mass spec is achieved
using calibrator and an internal standard.

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The acceptability of each run is evaluated
with different levels

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of controls.

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Collaborators and controls are samples
that have a known amount

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of the analyte added to a matrix
that matches the matrix of the patient.

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Samples.

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A fixed amount of an internal
standard is added

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to the collaborators
controls and patient samples

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to allow the amount of analyte
in a patient sample to be quantified.

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The internal standard is typically either
an isotope

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labeled version of the analyte,
or another compound

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with very similar chemical characteristics
as the analyte.

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The internal standard helps
to account for day to day variations

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observed during the extraction
and analysis of the samples.

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The samples

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are further processed
using one of several techniques available.

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Labs determine the most suitable process
to use based on the chemistry

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and matrix of the analytes being tested.

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The sample of interest

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is extracted using column chemistry,
where impurities and other interfering

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substances are removed
from the original specimen.

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The specimen is transferred

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to a 96 well plate for the next step.

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The samples are
then loaded into an auto sampler

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for instrumental analysis.

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For clinical testing.

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There are three types of mass spec
analysis in common use.

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Liquid chromatography.

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Mass spec LCMs gas chromatography.

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Mass spec GCMs,

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and inductively coupled plasma
mass spec ICP.

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Mass.

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This video will show the LCMs workflow.

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The auto sampler

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introduces each sample
based on a list into the instrument,

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with an automated sample needle,
which is washed after each sample.

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The LCMs then further separates
the analyte from other sample constituents

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using liquid mobile

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phase and a stationary phase housed
within the analytical column.

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The analyte is introduced into a stream
of mobile phases in a specific amount

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that ensures the analyte adheres to the
stationary phase in the analytical column.

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The ratio of mobile phases is
then changed to release the analyte

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in a controlled manner,

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to separate it from any interferences
still present in the sample.

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The analyte is released from the column
and is carried by the mobile phases

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into the mass spec source.

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Here, the

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liquid stream flows into a nebulizer
where a carefully controlled

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combination of vacuum temperature,
gas flow, and electric voltages

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help vaporize the stream and introduce
a charge to the analyte of interest.

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This causes the solvent of the sample
to evaporate and the analyte

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to create an ion, which will then
enter the ion path of the instrument.

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Within a triple clad mass spec,
the molecular ions pass through a region

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where the analyte is separated further
from any mobile phase remnants.

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Next, the ion optics region helps

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focus the ion beam and three quadruples
Q and three.

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Further, select for the iron or analyte
of interest using electromagnetic fields,

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electric currents,
and an inert collision gas.

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Quadruples are the functional units
of a triple quad mass spectrometer.

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Each of these
is basically an electromagnet.

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In two planes.

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It creates a magnetic field that attracts
the ionized molecule to one of the poles,

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and then switches the plane and pole
to which the ion is attracted.

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The switching from pole to pole
is carefully controlled

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to cause the molecule to fly in a spiral
path down the middle of the quadrupole.

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The instrument is calibrated
to ensure the accurate selection

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of only the ions of interest
through the center of the quadrupole ions,

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with different mass to charge
ratios are filtered out

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due to the high rate of change
and the currents of the magnetic fields.

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Many different
analytes can be evaluated in the same run.

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Ions that

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make it through all three quadruples
reach the detector, which then amplifies

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the signal from the ions
to generate a voltage that is measured

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and recorded by the onboard computer.

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These voltages are then plotted on a graph
known as the Chromatogram.

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For each channel.

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Each channel represents
one precursor to product mass transition.

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These different transitions for each
analyte are referred

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to as quantifier
and qualifier ions or transitions.

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It is best practice to monitor two
diagnostic transitions for each analyte.

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The filtering, fragmenting, and filtering
again, is

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what makes the mass spec methods highly
specific and sensitive.

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Data analysts review
every run's curve controls

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and internal standards to ensure
all are within established parameters.

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All data analyzes are performed
in duplicate before releasing results

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to physicians.