Analysis of nucleic acids by on‐line liquid chromatography–Mass spectrometry
Authors: Huber Christian G. , Oberacher Herbert 
Published: August 2002

I.
Introduction
311

A. Nucleic Acid Structures and Properties
311

B. Principles of LC of Nucleic Acids
312

  1. Reversed‐Phase HPLC
312

  2. Ion‐Pair Reversed‐Phase HPLC
314

  3. Affinity Chromatography
314

C. Principles of ESI MS of Nucleic Acids
314

II.
On‐Line Hyphenation of LC and MS
315

A. Ionization Techniques for Nucleic Acids Operable Under Continuous Flow Conditions
315

B. Instrumental Aspects and Miniaturization
316

C. Optimization of Chromatographic and Mass Spectrometric Experimental Conditions
317

  1. Eluent Composition in Reversed‐Phase‐ and Ion‐Pair Reversed‐Phase HPLC‐ESI‐MS
317

  2. Concentration of Organic Solvent in the Electrosprayed Solution
319

  3. Optimal Experimental Conditions for Ion‐Pair, Reversed‐Phase HPLC‐ESI‐MS
321

  4. Eluent Composition in Affinity Chromatography‐ESI‐MS
321

  5. Manipulation of Charge‐State Distribution by Sheath Liquids
321

III.
Performance Characteristics of HPLC‐ESI‐MS for Nucleic Acids
322

A. Separation Efficiency, Size Range, and Mass Accuracy
322

B. Detection Limits for Single‐ and Double‐Stranded Nucleic Acids
324

IV.
Applications
324

A. Synthetic Oligodeoxynucleotides
324

B. LC‐Tandem MS for Sequence Determination
325

C. Antisense Oligonucleotides and Oligonucleotide Metabolism
328

D. LC‐Tandem MS for Identification of DNA Adducts
331

E. Ribonucleic Acids
332

F. Products of PCR
333

G. Double‐Stranded DNA Restriction Fragments
336

H. Detection of Mutations by LC‐MS
338

References
339

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High‐performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single‐ and double‐stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on‐line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion‐pair reversed‐phase HPLC and electrospray ionization mass spectrometry (ESI‐MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC‐ESI‐MS include the characterization of oligodeoxynucleotides synthesized by solid‐phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double‐stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids. © 2002 Wiley Periodicals, Inc., Mass Spec Rev 20:310–343, 2001; Published online in Wiley Interscience (www.interscience.wiley.com). DOI 10.1002/mas.10011