PHARMACEUTICAL & BIOTECHNOLOGY
APPLICATIONS
ROCKY MOUNTAIN INSTRUMENTAL
LABORATORIES
Mass spectrometry, especially HPLC/MS/MS, is an important and quite useful technique for the detection, identification, quantitation and analysis of small pharmaceutical molecules, peptides, proteins, and oligonucleotides and their metabolites and degradants. With our SciEx triple quadrupole instruments with ElectroSpray (ESI) and NanoSpray ionization (NSI), we can be especially effective in the analysis of small quantities of proteins and protein digests. We also offer APCI (Atmospheric Pressure Chemical Ionization) as this technique sometimes gives better sensitivity than does ESI or NSI, especially for small, non-polar molecules, such as steroids.
Mass spectrometry also can be used to assist in the
identification of degradation products or contaminants in pharmaceutical
products. This may involve the forced degradation of API or finished
products. Note that, at times, excipients may be incompatible with
otherwise stable actives.
MASS SPECTROMETRY TECHNOLOGY:
There are several common
modes of obtaining mass spectra. These include: Time-of-flight (TOF), quadrupole,
ion trap, magnetic sector, and combinations of these. Ionization techniques
commonly used in biotechnology and pharmaceutical analysis for non-volatile
samples include Matrix-Assisted Laser Desorption/Ionization (MALDI), Electrospray
Ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI) and Fast
Atom Bombardment (FAB). Each technique has its own set of advantages and
disadvantages. That is, no one technique will solve all problems.
Tandem quadrupole mass spectrometry
(MS/MS) is especially useful for the analysis of pharmaceuticals in biological
samples because it allows for a very large improvement in the signal to
noise ratio (S/N) by effectively excluding interfering substances from
the analysis. Similarly, the use of both CI (chemical ionization)
and EI (electron ionization) can increase specificity and sensitivity.
This is why we are able to quantitate many molecules down to the 50pg/mL
level in plasma and solid tissues. We
offer both HPLC/MS/MS and GC/MS/MS. We have established even smaller
LOD and LOQ for certain molecules.
Proteins,
peptides, and oligonucleotides may be analyzed by the use of Electrospray
Ionization (ESI) of effluents from a liquid chromatograph
or capillary electrophoresis system. This is because it is possible to
generate an envelope of multiply charged species whose m/z is within the
mass range of the spectrometer (generally up to 3000 amu). Deamidation,
iso-aspartate formation, and other post-translational modifications and
the polymer sequence can be identified in proteins and peptides with relative
ease and excellent mass accuracy (1, 2). Oligonucleotide
molecular weight determination and sequence confirmation is performed by
very similar means, except that the sample preparation is a bit different
and negative ion mode is used (3). Because we use quadrupole MS/MS, we
are able to perform parent ion scans, which often improves S/N significantly
(5).
MALDI-TOF is often useful for the same work as is ESI with a quadrupole mass spectrometer, except that the multiply charged ionization is not used. The differences between the techniques are described more fully in reference (4). Other information can be obtained from the American Society for Mass Spectrometry (ASMS) and from manufacturers of the instrumentation. MS data libraries can be obtained from the NIST and Palisade Corp. The Journal of Mass Spectrometry is an excellent source of practical information and tutorials. A very good tutorial on peptide sequencing is to be found at Ionsource. Our RML home page lists many manufacturers and excellent sources of data and scientific information.
There now are several instrument manufacturers who sell combination quadrupole - TOF mass spectrometers for use with HPLC. They can be enormously useful in some cases, but tend to make conventional triple quadrupoles seem to be inexpensive.
On our home page, we list many sources of information regarding proteomics, protein analysis, and mass spectrometry.
REFERENCES:
1. D. R. Marshak (ed.) "Techniques in Protein Chemistry" This is a series of excellent books which contain papers presented at the Protein Society Symposia. Published by Academic Press. Very inexpensive, remarkably so for the high quality of information provided. The earlier volumes have information which is especially useful to anyone just starting in modern protein and peptide analysis. See volumes V and VI, particularly. (800)-782-4479
2. D. W. Aswad "Deamidation and Iso-aspartate Formation in Peptides and Proteins" CRC Press (1995).
3. J. Stults and J. Marsters "Improved Electrospray Ionization of Synthetic Oligonucleotides" Rapid Comm. in Mass Spec. 5, 359-363 (1991).
4. G. Suizdak "Mass Spectrometry for Biotechnology" Academic Press (1996). This is an excellent introduction to mass spectrometry, in general, and to the biotechnology applications, in particular, and is quite inexpensive.
5. G. Neubauer and M. Mann "Parent Ion Scans of Large Molecules" J. Mass Spectrometry 32 94-98 (1997).
6. R. K. Lantz and P. L. Sulik, "Liquid Chromatography - Mass Spectrometry: A Users Perspective" European Pharmaceutical Contractor, October, 1998.
7. R. B. Cole, ed. "Electrospray Ionization Mass Spectrometry: Fundamentals, Instrumentation, and Applications" Wiley (1997). Superb text for both the novice and the expert.
8. Danicik, V. et al, "De Novo Peptide Sequencing via Tandem Mass Spectrometry. J. Computational Biology , 327-342 (1999).
For additional information:
970-266-8108
888-985-8108 303-530-1169
Rocky Mountain Instrumental Laboratories, Inc.
108 Coronado Ct.
Ft. Collins, CO 80525
LAST UPDATE: 28 JANUARY 2009