LC/MS Emerges as QC/QA Standard

N/As engineers who remember room-sized research mass spectrometers requiring dedicated operators will be pleased to learn that many of today's instruments are benchtop- or workstation-sized. Even better, the instruments are inexpensive enough for routine quality assurance/quality control (QA/QC) in most pharmaceutical processes. Modern mass spectrometers interface easily with liquid chromatography systems to deliver fast, accurate identification of all components in complex process streams with a single injection.

The top molecular targets for liquid chromatography/mass spectrometry (LC/MS) systems are small molecule drugs, peptides, proteins, oligonucleotides, and DNA/RNA. Process and QA/QC engineers can now use these versatile instruments as easily as use liquid or gas chromatography. In fact, most manufacturers of benchtop LC/MS systems claim that users can be up and running within a week.

Key Applications
QA/QC engineers can use LC/MS for most in-process chemical analyses, including:

• Reaction monitoring for both small-molecule and biopharmaceutical processes
• Methods development for bulk chemicals, biological media, formulations
• Impurity profiling and characterization: identity, isolation, quantitation
• Dissolution testing
  
• Preformulation analysis, especially stability testing, evaluation of simulated formulations, and degradation product analysis
• Determination of pyrogens, DNA/RNA, and other impurities in cell-derived pharmaceuticals
  

Not Just Molecular Ions
The molecular ion peak, M+, the most fundamental piece of information from a mass spectrum, was once the only reason MS samples were run. With the rising complexity of small-molecule drugs - not to mention the importance of biomolecule drugs - fragment ions loom large. These enable QA/QC engineers to determine unequivocally that a particular LC peak and M+ peak belong together. And, modern LC/MS instruments are sensitive to tenths of a molecular weight unit, which through measurement of isotopic ratios allows precise determination of subtle changes in molecular structure, common in pharmaceutical batches.

Key Players
Recent LC/MS product introductions, plus the acquisition of PerSeptive Biosystems (Framingham, Mass.) by

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(Norwalk, Conn.) last month, underscored the emphasis instrument companies are placing on LC/MS. Drawing on PerSeptive's separations technology and Perkin-Elmer's experience in LC/MS, the two companies plan to create the first integrated platform of analytical products for compressing pharmaceutical time to market. PerSeptive's Mariner benchtop time-of-flight (TOF) LC/MS instrument, introduced at PittCon last year, generates high-resolution mass spectra on par with those from much larger, more expensive instruments. PerSeptive also markets the DE series of LC/MS systems based on "delayed extraction," a technique that introduces a time delay between ionization and extraction, resulting in higher signal-to-noise and, the company claims, between five- and ten-fold improvements in mass resolution.

Here's a rundown of other recent, significant product introductions:

Waters Corp.(Milford, Mass.). The Integrity System combines photodiode array and mass spectral detection in a unit that identifies both known and unknown compounds. Integrity operates at standard HPLC flow rates; cleaning and re-calibration take less than one hour. Waters' ThermaBeam mass detector uses a nebulizer to vaporize chromatography eluant and analyzes molecules by electron impact ionization. Waters' Alliance LC/MS (see Figure 1) features the Platform LC Detector from Micromass Ltd. (Manchester, U.K.), a company acquired by Waters late in 1997. The Platform LC uses atmospheric pressure ionization with Micromass's CrossFlow technology as an option. Atmospheric ionization generates pseudomolecular ions, from which the molecular weight of a desired peak is deduced.

Waters' Alliance LC/MS

Micromass will begin marketing its Quattro LC around the time of PittCon '98. The Quattro is expected to feature a new sampling technique, "Zspray," which avoids contamination while delivering enhanced signal to noise. In Zspray unwanted components of the sample spray, such as droplets and nonvolatile materials, are vented before they hit the detector. Quattro may be combined with a large number of third-party analytical systems and delivery equipment, including solvent delivery systems, autosamplers, and extractors.

Finnigan's (San Jose, Calif.) top of the line instrument, the LCQ MSⁿ detector (Figure 2), works through atmospheric pressure ionization, electrospray ionization, or atmospheric pressure chemical ionization probes. Unlike other LC/MS interfaces, which operate either at slow or fast flow rates but not in between, the MSⁿ operates at flows ranging from 1 mL/min to 1 mL/min, without splitting, for the electrospray probe and from 500 mL/min to 1 mL/min for the other probes.

Finnigan's LCQ LC/MS

JEOL Ltd. (Peabody, Mass.) has designed a new ion source and spectrometer for its LCmate (Figure 3), a fully automated, benchtop, double-focusing LC/MS. Atmospheric pressure ionization comes standard with the LCmate, with optional electrospray ionization. Since electrospray technology generates multiply-charged ions, the LCmate is ideal for peptides and proteins, as well as smaller drug molecules.

JEOL's LCMate LC/MS

Sensar Corp. (Provo, Utah), which uses time-of-flight MS technology developed at Brigham Young University, offers the Jaguar line of mass detectors for use with LC systems. Jaguar primarily uses electrospray, although other ionization techniques also work. In the Jaguar ions expand supersonically into the vacuum system through a nozzle skimmer, and focus into a tight beam by collisional cooling. Sensar also offers the Simul-Pulse recorder, which addresses critical data acquisition problems of modern LC/MS instruments, namely very large data files.

The API 165 single quadrupole LC/MS system from Perkin-Elmer Sciex Instruments (PE Sciex; Foster City, Calif.), uses a patented Curtain Gas interface which dads stability and ruggedness to MS analysis. Curtain Gas desolvates ions and protects the ion path from contamination, even with dirty process samples. The API 165 uses PE Sciex's MassChrom software, a powerful, easy-to-use package controlling all functions of the MS, LC pumps, and autosampler.

MS Definitions

Atmospheric pressure chemical ionization:

Consists of an aerosol generator, a desolvator (usually heat), and a high-voltage discharge needle. Ionization involves aqueous protonation, which means molecules must have a higher affinity for protons than water. Back

Electrospray:

A technique which allows any ion, of any size, to be transported intact into the gas phase for MS analysis. Back

Time of flight (TOF):

The simplest type of MS analyzer. TOF systems require a pulsed ion source, a flight tube, and an ion detector. The mass of an ion is related to the time it takes an ion to travel through the flight tube to the detector. Back

Quadrupole mass analysis:

A type of mass filter, consisting of four electrically charged rods, which samples ions continuously (Figure 4).

Schematic of the quadrupole analysis Back

Leading LC/MS Manufacturers
Finnigan, San Jose, CA. Tel: 408-433-4800.
JEOL USA, Peabody, MA. Tel: 508-535-5900.
MicroMass Ltd., Beverly, MA. Tel: 508-524-8200.
PerSeptive Biosystems, Inc., Framingham, MA. Tel: 508-383-7700.
Perkin-Elmer Sciex, Norwalk, CT. Tel: 203-762-1000.
Sensar, Provo, UT. Tel: 801-343-3617.
Waters Corp., Milford, MA. Tel: 508-872-2000.

by Angelo DePalma