Analysis of nucleic acids by on‐line liquid chromatography–Mass spectrometry

I. Introduction 311A. Nucleic Acid Structures and Properties 311B. Principles of LC of Nucleic Acids 312  1. Reversed‐Phase HPLC 312  2. Ion‐Pair Reversed‐Phase HPLC 314  3. Affinity Chromatography 314C. Principles of ESI MS of Nucleic Acids 314II. On‐Line Hyphenation of LC and MS 315A. Ionization Techniques for Nucleic Acids Operable Under Continuous Flow Conditions 315B. Instrumental Aspects and Miniaturization 316C. 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 321III. Performance Characteristics of HPLC‐ESI‐MS for Nucleic Acids 322A. Separation Efficiency, Size Range, and Mass Accuracy 322B. Detection Limits for Single‐ and Double‐Stranded Nucleic Acids 324IV. Applications 324A. Synthetic Oligodeoxynucleotides 324B. LC‐Tandem MS for Sequence Determination 325C. Antisense Oligonucleotides and Oligonucleotide Metabolism 328D. LC‐Tandem MS for Identification of DNA Adducts 331E. Ribonucleic Acids 332F. Products of PCR 333G. Double‐Stranded DNA Restriction Fragments 336H. Detection of Mutations by LC‐MS 338 References 339The 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