Lakhasly

High-risk babies or children from regional hospitals are routinely referred to the Metabolic Division of Child Health Department at SQUH. A total of 1100 patients were referred and investigated

for inborn errors of metabolism during a period of 10 years (1998-2008). Whole blood samples were drawn by heel prick or venipuncture from high-risk babies and spotted on Guthrie filter cards No. 903 Whatman paper (Schleicher and Schuell, Dassel, Germany). Four spots of whole blood were collected and allowed to dry at room temperature for at least three hours. Samples were then sent to the laboratory in plastic envelopes and stored at 4°C until analyzed.
In an attempt to reveal the pattern of distribution of IEMs in Oman, the geographical location as well as the ethnic and tribal origin of parents was examined. The Omani population was divided into three ethnic groups: Arabs of the north, Arabs of the south (Dhofari), and Omanis of Asian descent. Northern Arabs included all Arab tribes occupying the northern parts of Oman. Southern Arabs included all the Arabs from Dhofar and southern parts of Oman. Omanis of Asian Origin included migrants from Iran, India, Pakistan and Afghanistan.
Mass spectrometry is a technique used for identifying and quantifying analytes based on their molecular mass and charge. For analysis by a mass spectrometer, the analyte of interest must be converted into ions. Ions are separated by electromagnetic fields, detected by a quantitative method and processed into mass spectra (Fig. 1) according to their mass to charge ratio (m/z). Therefore, the main four components of MS instrument are sample inlet, ion source, mass analyzer, and a detector. The vertical axis represents ion intensity expressed as a % of the largest peak in the spectrum. Internal standards are represented with a small superscript star

In Electrospray-ionization tandem mass spectrometry (ESI- MS/MS), a sample is first dissolved in a mixture of water and or- ganic solvent (e.g., acetonatrile). Then, the extract is derivatized with butanol to form butyl esters of amino acids, organic acids, and fatty acids. As the mass spectrometer cannot detect uncharged molecule, the analyte must be ionized before it is introduced to the first MS (MS 1). Ionization of the derivatized analyte is achieved by electro- spray ionization in which the sample effluent is passed through a small capillary to which a high voltage has been applied. The ioniza- tion process involves the transfer of a charge to the solvent droplets, evaporation of the solvent, and finally, production of positively and negatively charged ions. This vaporized and ionized mixture enters the MS 1, which functions as a separating device and allows only the ion(s) of interest to pass through. Ions passing through the MS 1 are called precursor or parent ions. Once the ion passes through the MS 1, it enters the collision cell where fragmentation takes place.
Fragmentation is typically achieved by introduction of an inert gas such as argon into the collision cell. The fragments generated in the collision cell are called product or daughter ions. In the second mass spectrometer (MS2), the product or daughter ions are ana- lyzed in the same manner as in the first mass spectrometer. Products or daughter ions in the MS2 can be correlated with the parents ions produced in the MS 1. This process enables unique mass spectrom- etry such as precursor ion scans and neutral loss scans. Since certain compound classes share common fragment ions of neutral fragment molecules, a special analysis can be set up to only detect precursor ions with a particular fragment. This results in an ability to measure only a particular chemical class or subset of molecules without de- tecting the hundreds of molecules that are not of interest.
ESI-MS/MS was carried out on a Quattro Micro (Manchester, UK), triple quadrupole spectrometer, equipped with a Waters Alliance 2790 HPLC pump, with minor modification of methods developed by Chance et al.1-2 and Rashed et al.3
A 3 mm disk from a selected spot from the Guthrie card was punched out using a standard hole punch in each of a 96-well polypropylene microtitre plate. The sample in the blood spot punch in each well was then extracted by adding 100 μl of isotope- labeled internal standard solution of amino acids and acylcarnitines (Cambridge Isotope Labs, Andover, MA, USA) and incubated at room temperature for 30 minutes. The extracted sample was then transferred to another plate. The solvent was gently evaporated and 60 μl of butanolic HCl was added. The plate was sealed with special heat-resistant tape and heated at 65oC for 15 minutes. The solvent was evaporated again and the residue was finally reconstituted in 100 μl of the mobile phase (acetonatrile/water, 80/20 v/v) and fed into the system through the HPLC pump.
Neolynx software (Neolynx Inc., Glendale, California, USA) was used for processing MS/MS data. Neolynx operates by calculating the ratio of the intensity of the mass spectral peak for the analyte to the intensity of the mass spectral peak for a pre-defined internal standard. If the concentration of the internal standard is known then the concentration of the analyte may be calculated by multiplying the peak intensity ratio by the concentration of the internal standard. Samples were analyzed in duplicates in the same analytical run. If a sample is flagged as abnormal, a repeat sample for MS/MS analysis was requested. If the second sample agreed with the first sample result, the diagnosis was then confirmed by urine organic acids analysis, plasma amino acids analysis, or direct enzyme assay; all analyzed by Biomnis laboratory, France (BIOMNIS Specialised Medical Pathology, International Division, Lyon Cedex 7 – FRANCE).
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