newborn mass screening for classical 21-hydroxylase deficiency ( 21ohd ) by measurements of the 17ohp level in filter paper blood using an enzyme - linked immunosorbent assay has been used in japan with high reliability ( 1 ) . definitive diagnosis of classical 21ohd is , however , not easy , partly because immunochemical measurement of the serum 17ohp level is problematic due to the cross - reactivity of antibodies with a large number of fetal adrenal steroids ( 2 ) . theoretically , measurement of the serum 17ohp level by non - immunochemical stable isotope dilution - gas chromatography / mass spectrometry ( sid - gc / ms ) is more accurate than measurement by immunochemical methods ( 3,4,5 ) . however , few reference values for the serum 17ohp level in newborn infants using sid - gc / ms have been reported ( 6 ) . furthermore , no studies comparing measurement of the 17ohp level by sid - gc / ms with other immunochemical methods have been reported . the aims of this study were 1 ) to establish the reference values for the serum 17ohp level in japanese newborn infants using sid - gc / ms and 2 ) to compare the levels of serum 17ohp determined using sid - gc / ms with those determined by radioimmunoassay ( ria ) in newborn infants . the first subjects were used to establish reference values by sid - gc / ms and included 57 healthy full - term japanese newborn infants ( 35 d of age , 32 boys and 25 girls , 3742 wk gestation , birth weight 2,5003,824 g ) . the second subjects were used for comparison of sid - gc / ms with ria and included 1 ) 27 healthy full - term japanese newborn infants ( 36 d of age , 16 boys and 11 girls , 3741 wk gestation , birth weight 2,6034,162 g ) and 2 ) two subjects with neonatal transient hyper 17ohpnemia ; these two subjects were 16 and 27 d of age , respectively . diagnosis of neonatal transient hyper 17ohpnemia was based on the combination of the following ; 1 . no clinical signs and symptoms of classical 21ohd , 2 . normal serum na and k level , 3 normal serum 17ohp level later in life . the parent(s ) of the subjects gave informed consent . the 17-hydroxyprogesterone ( 17ohp ) was purchased from sigma - aldrich ( st . louis , mo , u.s.a . ) , and [ 11,11,12,12-d4]-17ohp was synthesized by us ( n.y . ) as described previously ( 7 ) . heptafluoro - n - butyric anhydride ( hfba ) was obtained from gl sciences ( tokyo , japan ) . dichloromethane , isooctane , cyclohexane , acetone and ethanol were of analytical reagent grade and were used without further purification . a dpc 17-oh progesterone kit was purchased from mitsubishi kagaku iatron ( tokyo , japan ) . the serum 17ohp level ( ng / ml ) was measured by sid - gc / ms using 100 l of serum . in brief , the method consisted of extraction , derivatization , and gc / ms analysis . the procedures were as follows ; 1 . five ng of [ 11,11,12,12-d4]-17ohp , as an internal standard , in 5 l of ethanol and 1 ml of distilled water was added to 100 l of neonate serum or 17ohp standard solution ( 0.05 , 0.1 , 0.2 , 0.5 , 1 , 2 , 5 , 10 , or 20 ng / ml in ethanol ) . steroids were extracted with 3 ml of dichloromethane / isooctane ( 1:2 v / v ) . one hundred microliters of acetone and 25 l of hfba were added to the mixture . gc / ms analysis was performed on an hp 5890ii gc with an hp fused silica column ( 25 m ) coupled to an hp 5971 ms ( agilent technologies , palo alto , ca , u.s.a . ) . the temperature programming was 100 c ( 2 min ) , 100230 c ( 30 c / min ) , 230290 c ( 3 c / min ) and 290 c ( 10 min ) . selected ion monitoring ( sim ) analysis was performed with two characteristic mass ions selected for each steroid ( 465.3 and 369.2 m / z for 17ohp ; 469.3 and 373.2 m / z d4 - 17ohp ) . the turnaround time of the assay was 12 h. the serum 17ohp level was also measured by i - ria ( dpc 17-oh progesterone kit ) using 25 l of serum after ether extraction , according to manufacturer s instructions . the sensitivity was 0.02 ng / tube ( 0.2 ng / ml in serum ) . ( coefficient of variation ) was 3% ( n=5 , 12.8 ng / ml ) . linearity was observed from 0.05 ng to 20 ng ( 0.5200 ng / ml in serum ) . the reference values of serum 17ohp in newborn infants determined by sid - gc / ms ranged from 0.3 to 1.5 ng / ml with a median of 0.6 ng / ml . out of 27 healthy full - term newborn infants , the serum 17ohp level determined by sid - gc / ms was lower in one case ( 1.5 ng / ml ) compared with the level determined by ria after extraction ( 3.6 ng / ml ) . in the other 26 subjects , the serum 17ohp levels determined by sid - gc / ms were almost the same as those by determined by ria after extraction ( fig . 1 correlation between the levels of 17ohp determined by sid - gc / ms and ria after extraction . the asterisk ( * ) indicates the case in which the 17ohp level determined by sid - gc / ms was lower than the level determined by ria after extraction . ) . two subjects with neonatal transient hyper 17ohpnemia had lower serum 17ohp levels determined by sid - gc / ms ( 3.1 and 1.1 ng / ml ) compared with the levels determined by ria after extraction ( 9.8 and 4.2 ng / ml ) . correlation between the levels of 17ohp determined by sid - gc / ms and ria after extraction . the asterisk ( * ) indicates the case in which the 17ohp level determined by sid - gc / ms was lower than the level determined by ria after extraction . the sensitivity was 0.02 ng / tube ( 0.2 ng / ml in serum ) . ( coefficient of variation ) was 3% ( n=5 , 12.8 ng / ml ) . linearity was observed from 0.05 ng to 20 ng ( 0.5200 ng / ml in serum ) . the reference values of serum 17ohp in newborn infants determined by sid - gc / ms ranged from 0.3 to 1.5 ng / ml with a median of 0.6 ng / ml . out of 27 healthy full - term newborn infants , the serum 17ohp level determined by sid - gc / ms was lower in one case ( 1.5 ng / ml ) compared with the level determined by ria after extraction ( 3.6 ng / ml ) . in the other 26 subjects , the serum 17ohp levels determined by sid - gc / ms were almost the same as those by determined by ria after extraction ( fig . 1 correlation between the levels of 17ohp determined by sid - gc / ms and ria after extraction . the asterisk ( * ) indicates the case in which the 17ohp level determined by sid - gc / ms was lower than the level determined by ria after extraction . ) . two subjects with neonatal transient hyper 17ohpnemia had lower serum 17ohp levels determined by sid - gc / ms ( 3.1 and 1.1 ng / ml ) compared with the levels determined by ria after extraction ( 9.8 and 4.2 ng / ml ) . correlation between the levels of 17ohp determined by sid - gc / ms and ria after extraction . the asterisk ( * ) indicates the case in which the 17ohp level determined by sid - gc / ms was lower than the level determined by ria after extraction . we have successfully established reference values for the serum 17ohp level in newborn infants by sid - gc / ms , 0.3 to 1.5 ng / ml with a median of 0.6 of ng / ml ; this was similar to the values reported by wudy et al . using the same methods , 0.5 to 4.3 ng / ml with a median of 1.8 ng / ml ( 6 ) . it is noteworthy that the basic properties of our method were good judging from the recovery rate , sensitivity , intra - assay c.v . , and first , measurement of the serum 17ohp level by sid - gc / ms was reliable , judging from the method s good basic properties , such as the recovery rate , sensitivity , intra - assay c.v . , and linearity . second , we used 100 l of serum for 17ohp measurement by sid - gc / ms , while we used 25 l for determination of the 17ohp level by ria . the larger volume required for measurement might be a weak point of sid - gc / ms , however the turnaround time of the assay is 12 h. third and most importantly , the serum 17ohp levels determined by sid - gc / ms were lower than those by ria in one control subject and two subjects with neonatal transient hyper 17ohpnemia . theoretically , determination of the serum 17ohp level by sid - gc / ms is more accurate . namely , the serum 17ohp level determined by ria was higher than the true value , probably because fetal adrenal steroids could cross - react with the antibody used in the ria . in preterm newborn infants , who have more residual fetal adrenal gland than full - term newborn infants , sid - gc / ms would be a good candidate for accurate measurement of the serum 17ohp level . fourth , measurement of the serum 17ohp level by sid - gc / ms may be more useful than ria for definitive diagnosis of classical 21-hydrozylase deficiency ( 21ohd ) in newborn infants . we believe that these reference values of serum 17ohp are relevant to definitive diagnosis of 21ohd as well as other adrenal disorders in newborn infants , such as p450 oxidoreductase deficiency ( 8 , 9 ) . in summary , we have established reference values for the serum 17ohp level in newborn infants by sid - gc / ms . we are currently addressing the clinical utility of our method in differential diagnosis of 21ohd in newborn infants , who show elevated 17ohp level in filter paper blood as determined by elisa in neonatal screening .
immunochemical measurement of serum 17-hydroxyprogesterone ( 17ohp ) , the most important parameter for diagnosis of classical 21-hydroxylase deficiency ( 21ohd ) in newborn infants , is known to be inaccurate due to the cross - reactivity of antibodies with a large quantity of fetal adrenal steroids . 
 the aims of this study were 1 ) to establish reference values for the serum 17ohp level in japanese newborn infants using non - immunochemical stable isotope dilution gas chromatography / mass spectrometry ( sid - gc / ms ) and 2 ) to compare the serum 17ohp levels determined by sid - gc / ms with those determined by radioimmunoassay ( ria ) . 
 the first study subjects were used for determination of reference values and included 57 healthy full - term newborn infants ( 45 d of age ) . 
 the second study subjects were used for comparison of sid - gc / ms with ria and included 27 healthy full - term newborn infants ( 36 d of age ) and two subjects with neonatal transient hyper 17ohpnemia ; these two subjects were 16 and 27 d of age , respectively . in the first study subjects , 
 the intra - assay coefficient of variation for sid - gc / ms was 3% ( n=5 ) , the recovery rate was 98% , the sensitivity was 0.2 ng / ml , and the range of linearity was 0.5200 ng / ml . the reference values for the serum 17ohp level determined by sid - gc / ms ranged from 0.31.5 ( 0.6 ) ( ng / ml ) ( median ) . in the second study subjects , 
 the serum 17ohp levels determined by sid - gc / ms were lower in one of the 27 subjects and both of the two subjects with neonatal transient hyper 17ohpnemia compared with the levels determined by ria . 
 measurement of the serum 17ohp level using sid - gc / ms may be clinically useful for definitive diagnosis of classical 21ohd in newborn infants .